W: .'«^M-^ MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME (DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE) AND THE POST-PREPHENATE PATHWAY ENZYMES NEEDED FOR PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP, BY CAROL ANN BONNER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1994 ACKNOWLEDGEMENTS Many thanks to Roy A. Jensen for the inspiration and guidance throughout my studies for this dissertation. I thank, also, ray committee members. Dr. Richard P. Boyce from the Department of Biochemistry and Molecular Biology; and frora the Department of Microbiology and Cell Science, Dr. Lonnie 0. Ingram, Dr. Robert R. Schmidt and Dr. Keelnatham T. Shanmugam for their support and encouragement in completion of this dissertation. Special thanks to all the members of our lab who have been a great source of help to me during these studies. Firstly, to Randy Fischer and Premila Rao, my most grateful thanks for their help and encouragement in so many ways from the very beginning of my studies. Thanks to Astride Rodrigues, Jackie Miller, Michelle Miller for all the work in the tissue culture lab. Most special thanks to Tianhui Xia, Genshi Zhao, Prem Subramaniara, Wei Gu and Jain Song for all the discussion and work we have done together. I would also like to thank the other members of the microbiology and cell science department who have helped me with their knowledge and advice, especially Philip Miller, for all the information and insight that he gave me with respect to the molecular biology portion of my dissertation. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF FIGURES V LIST OF TABLES viii ABBREVIATIONS ix ABSTRACT xi CHAPTERS I LITERATURE REVIEW AND RATIONALE 1 Aromatic Amino Acid Biosynthesis in Plants.. 1 Rational for Focus upon DQT and SDH 8 Shikimate biosynthesis and quinate catabolism 9 Multiple Species of Shikimate Dehydrogenase. 12 The Post-Prephenate Pathway in Plants 16 II MATERIALS AND METHODS 19 Organisms and Extract Preparation 19 Enzyme Assays 23 Separation of Pathway Enzymes by Chromatography 27 Purification of Pathway Proteins 29 Polyacrylamide Gel Electrophoresis 33 Kinetic Values 34 Inhibition studies 35 Antibodies 3 5 cDNA cloning and sequencing 3 7 Biochemicals 43 III PHYSIOLOGICAL PROFILE OF AROMATIC PATHWAY ENZYMES IN PLANTS 45 Results 45 Discussion 59 iii IV PURIFICATION OF PROTEINS FROM THE AROMATIC AMINO ACID PATHWAY 62 Results 62 Discussion 53 V PROPERTIES OF THE S-PROTEINS 76 Results 76 Discussion 104 VI SPECIFIC ANTIBODY TO THE BIFUNCTIONAL S- PROTEIN AND TO TWO POST-PREPHENATE PROTEINS 108 Results 108 Discussion 116 VII CLONING cDNAS ENCODING THE BIFUNCTIONAL S-PROTEIN, ADH AND PAT 118 Results 118 Discussion 132 SUMMARY 148 REFERENCES 14 9 BIOGRAPHICAL SKETCH 157 IV LIST OF FIGURES Figure Page 1-1 Aromatic amino acid biosynthetic pathway 3 1-2 Multiple compartment at ion of pathways in plant cells 7 1-3 Quinate catabolism and aromatic amino acid biosynthesis 11 3-1 DEAE-cellulose chromatography of aromatic pathway enzymes from N. silvestris 48 3-2 DEAE- Cellulose chromatography of aromatic pathway enzymes from C. sorokiniana 51 3-3 Temperature optimum for activity of prephenate aminotransferase in Chlorella sorokiniana 52 3-4 Growth curve of N. silvestris in suspension cultures 56 3-5 Arogenate dehydratase activities followed throughout a growth curve of N. silvestris 58 4-1 Elution profiles of SP-I and SP-II from Celite 545 column chromatography 64 4-2 Elution profiles of SP-I and SP-II from DEAE- cellulose chromatography 67 4-3 Elution profiles of S-proteins from an NADP* specific affinity column 70 4-4 Celite 545 column chromatography overlap of arogenate dehydrogenase and SP-II 72 4-5 Hydroxyl apatite column chromatography separation of arogenate dehydrogenase and SP-II 73 5-1 Elution profile of SDH activities from a Celite 545 column run with a shallow gradient 78 5-2 Molecular weight determinations by SDS PAGE and by silver stain gels of the two S-proteins 81 V 5-3 Gel filtration chromatography of S-proteins 83 5-4 Optimal PAGE activity staining for SDH in crude extract 86 5-5 PAGE activity staining of purified SI- and S2- proteins 88 5-6 S-proteins pH optima of optima for activity 90 5-7 Temperature optima for S-proteins 93 5-8 Thermal inactivation of S-proteins 95 5-9 Substrate protection against thermal inactivation of S-proteins 97 5-10 Saturation curves and double reciprocal plots for SP-1 100 5-11 Saturation curves and double reciprocal plots for SP-II 102 6-1 Precipitation of S-proteins with SP-I specific antibody on Ouchterlony plates 110 6-2 Effect of SP-I specific antibody on SP-I and SP-II activities 113 6-3 Comparison of Western blot and activity stained gels 115 7-1 Agarose gels of cDNA encoding aromatic pathway proteins 121 7-2 Preliminary alignment of translated sequences of clone SP3 with established DQT sequences 124 7-3 Sequencing strategy for cDNA encoding the bifunctional protein 126 7-4 Agarose gels of SP3 cDNA fragments during stages of subcloning 128 7-5 Nucleotide and deduced amino acid sequence of cDNA coding for the bifunctional S-protein 131 7-6 Multiple amino acid alignment of the AroD domain of AroD«E with its homologues 135 7-7 Dendrogram of dehydroquinase homologues 137 VI 7-8 Multiple amino acid alignment of the shikimate dehydrogenase domain of AroD«E and its homologues 141 7-9 Dendrogram for shikimate dehydrogenase homologues 143 Vll LIST OF TABLES Table Page 1-1 Molecular mass and Km values for DQT and SDH 15 3-1 Specific activities from N. silvestris and C. sorokiniana aromatic pathway enzymes 46 3-2 Prephenate aminotransferase amino acid donor specificity in C. sorokiniana 53 3-3 Prephenate aminotransferase activity after thermal treatment in C. sorokiniana 53 4-1 Purification of SP-I from N. silvestris suspension cultured cells 71 4-2 Purification of SP-II from N. silvestris suspension cultured cells 71 5-1 Kinetic parameters for SP-I and SP-II 103 5-2 Effect of PCMB on enzyme activity 104 6-1 S-protein antibody effects on N. silvestris and E. coli extracts 116 7-1 SDH/DQT activities in transformed aroD and aroE mutant strains of E. coli 122 7-2 Pairwise comparisons of S-protein with its homologues 144 7-3 NAD(P)H binding motif of enzymes 145 7-4 Transit peptides of aromatic pathway proteins and other plant proteins 143 Vlll ABBREVIATIONS ADH arogenate dehydrogenase ADT arogenate dehydratase AGN L- arogenate aKG a-ketoglutarate AMP ampicillin ANS anthranilate synthase ANS-1 aneuploid N. silvestris suspension cell line jSME beta mercaptoethanol BSA bovine serum albumin CHA chorismate CM chorismate mutase CM-52 carboxymethyl cellulose-52 cation exchange column CM- I chloroplast chorismate mutase CM- II cytosolic chorismate mutase DAHP 3-deoxy-D-araJblno-heptulosonate 7-phosphate DAHPS DAHP synthase, enzyme 1 of aromatic pathway DE52 (DEAE) diethylaminoethyl cellulose-52 anion exchange column DHQ dehydroquinate DHS dehydroshikimate DQT dehydroquinate dehydratase or dehydroquinase Ds-Co cytosolic DAHP synthase of plants Ds-Mn chloroplast DAHP synthase of plants DTT dithiothreitol E-cells cells of about two generations of exponential growth EDTA ethylenediaminetetraacetate EE-cells cells in continuous exponential growth EPPS (N- [2-Hydroxyethyl]piperazine-N' - [3- propanesulfonic acid] ;HPPS) EPSP 5 - enoipyruvyl shikimat e - 3 -phosphate E4P erythrose 4 -phosphate HA hydroxylapetite column HPLC high performance liquid chromatography HPP 4-hydroxyphenylpyruvate ICBR Interdisciplinary Center of Biotechnology Research IPTG isopropylthio-jS-D-galactoside KPO4 potassium phosphate buffer LB Luria-Bertaini medium LB-AMP LB plus ampicillin LES lag, exponential and stationary phases of growth MTT 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetra- zolium-bromide; (thiazolyl blue) NADP nicotinamide adenine dinucleotide NTPS nucleotide triphosphates ix OAA oxaloacetate OPA orthopthalaldyhyde PAGE polyacrylamide gel electrophoresis PAT prephenate aminotransferase PCA protocatechuic acid PCMB p-chloromercuribenzoate PDH prephenate dehydrogenase PDT prephenate dehydratase PEGgooo polyethylene glycol PEP phosphoenolpyruvate PLP pyridoxal 5 ' -phosphate PMS N-methyldibenzopyrazine raethylsulf ate (phenazine metasulfate) PMSF phenylmethylsulfonyl fluoride PPA prephenate PPY phenylpyruvate PCA protocatechuic acid QA quinic acid QDH quinate dehydrogenase QDT quinate dehydratase RE restriction enzymes SA specific activity S-protein shikimate-bifunctional DQT/SDH protein SDH shikimate dehydrogenase SDS sodium dodecyl sulfate SP-I (Sl-protein) bifunctional protein (DQT/SDH) , major peak from Celite 545 chromatography separation SP-II (S2 -protein) bifunctional protein (DQT/SDH) , minor peak from Celite 545 chromatography separation SHK shikimate Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MOLECULAR CHARACTERIZATION OF A BIFUNCTIONAL ENZYME (DEHYDROQUINATE DEHYDRATASE/SHIKIMATE DEHYDROGENASE) AND THE POST- PRE PHENATE PATHWAY ENZYMES NEEDED FOR PHENYLALANINE AND TYROSINE BIOSYNTHESIS IN NICOTIANA SPP, By Carol Ann Bonner April 1994 Chairman: Roy A. Jensen Major Department: Microbiology and Cell Science Two bifunctional proteins containing catalytic domains for dehydroquinate dehydratase (dehydroquinase) and shikimate dehydrogenase were separated from extracts of Nicotiana silvestris suspension cells by use of a decreasing ammonium sulfate gradient on a Celite 545 column. The two apparent isoenzymes, denoted as SP-I and SP-II, were purified 1000- and 800-fold, respectively. Higher molecular mass values were obtained for SP-II than for SP-I on the criteria of sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. Each protein exhibited multiple bands on SDS-PAGE and on native-PAGE monitored with a specific shikimate xi dehydrogenase activity stain. Temperature and pH optima for catalysis were similar for each protein, as were the temperatures of inactivation. However, Km values for shikimate were 0.80 and 0.36 mAT for SP-I and SP-II, respectively. Antibody raised against SP-I cross-reacted with SP-II, but not with Escherichia coli proteins possessing the corresponding activities. Antibody screening of a cDNA library of Nicotiana tabacum was used to isolate clones expressing the S -protein. Functional complementation of aroD and aroE E. coli auxotrophs transformed with plasmids carrying cloned cDNA were successful. Activities for each enzyme were 15 -fold greater in the transformed mutant strains than in the wild type strain of E. coli. Analysis of the amino acid sequence deduced from the cloned cDNA sequence revealed homology with the appropriate functional domains of the pentafunctional Arol protein of Saccharomyces cerevisiae and with the monofunctional AroD and AroE proteins of E. coli. The post-prephenate pathway enzymes were also studied in Nicotiana silvestris suspension cells. Levels of arogenate dehydratase activity changed throughout a growth cycle in suspension cells. Using specific antibodies, several putative cDNA clones encoding prephenate aminotransferase or arogenate dehydrogenase were isolated for further study. The base of comparative enzymology in higher plants for the aforementioned enzymes was extended to the unicellular alga, Chlorella sorokiniana . xii Prephenate aminotransferase of Chlorella exhibited the striking high- temperature activity optimum and specificity for prephenate that is typical of higher plants. Xlll CHAPTER I LITERATURE REVIEW AND RATIONALE Aromatic Amino Acid Biosynthesis in Plants In higher plants, the three aromatic amino acids (L- phenylalanine, L- tyrosine and L- tryptophan) are not only required for protein synthesis and primary metabolite synthesis (such as the hormone, indole acetic acid and the structural component of woody plants, lignin) , but also must be available as starting substrates for a vast array of secondary metabolites including alkaloids, coumarins, isoflavones, and tannins (2, 78, 79) . Intermediates of the pathway are also precursors for synthesis of other essential metabolites in plant cells. The vitamin-like derivatives, folic acid and ubiquinone, are synthesized from chorismate, the first branch point intermediate in the aromatic pathway. Protocatechuic acid is derived from the common pathway intermediate, dehydroshikimate (Fig. 1) . It has been estimated that up to 60% of plant carbon flows through the aromatic amino acid pathway (40) . Humans as well as other higher forms of life depend heavily on plants to provide the three essential aromatic amino acids, and knowledge of aromatic biosynthesis in higher plants is fundamentally Fig. 1-1. Aromatic amino acid biosynthetic pathway. The common portion of the biosynthetic pathway leading to the branch point intermediate, chorismate, begins with DAHP synthase [1] catalizing the condensation of erythrose 4 -phosphate and phosphoenolpyruvate and consists of seven steps. These steps consist of dehydroquinate synthase [2] , dehydroquinate dehydratase [3] , shikimate dehydrogenase [4] , shikimate kinase [5] , EPSP synthase [6] , and chorismate synthase [7] . Chorismate, at the branchpoint of the pathway is utilized in one direction to yield L-tryptophan (steps 8-12) and in another direction by chorismate mutase [13] to produce prephenate, another branch point intermediate. Organisms in nature use different routings from prephenate. Higher plant chloroplasts proceed through prephenate aminotransferase [14] and arogenate dehydratase [15] to form PHE, or through prephenate aminotransferase and arogenate dehydrogenase [16] to form TYR. In other organisms a second route proceeds through prephenate dehydratase [19] , then through aromatic (or PHE) aminotransferase [20] to synthesize PHE, or through prephenate dehydrogenase [17] and through aromatic (or TYR) aminotransferase [18] to synthesize TYR. MC-OM HC-OM I COOH COOM llANlll _^ t not t (111 t f COOM I e-o I CM, I ' Mo^ ^eooM MOC-M MCOM eMjO»Oj><2 MO COOM COO" COOH O^Nj^^OM 0^*V^^^S)H MO--Ky''>3M OH OH i- OH ill/*" f ^GLN ^a)— tOOM COOH COOM ^^ CH, -__<7, ^n] CHj „ -<^ €^ >r^^-<=-cooH H,OjW^Y'^o'«»eooH H^j»o-'^v-^OH 6" OH 6m Sd^ o CHj-e-cooH CM, -cm -COOH lui / / (191 -1201 '*'•'' » ll'"0 / NOOC. CMj-C-COOM I JH) 1,4 / (1S1 f HOOe,„^CHj.CM -COOH _-^ M) Om\ Om\ \ > \NAO*/NAOP* 1171 \ \NAO*/NAOP* I16I \ cm,-c-coom NH. y CMj-CN-COOH -(18)_!tL—HTY,;i 4 important. Figure 1 shows a composite schematic of the biosynthetic steps of the common portion of the pathway and of the three divergent amino acid branches. It has been established that a complete aromatic biosynthetic pathway is present in the chloroplasts of plant cells (2, 41) . The common portion of the pathway begins with the condensation of erythrose 4 -phosphate and phosphoenol pyruvate to form 3-deoxy-D-arajbino-heptulosonate 7-phosphate by DAHP synthase and continues with six more enzymatic steps to form CHA, the major branchpoint intermediate. From CHA, the pathway branches to form L- tryptophan via anthranilate synthase (ANS, enzyme 8) and to L-phenylalanine or L-tyrosine via chorismate mutase (CM, enzyme 13) . From prephenate, the product of the CM reaction, different pathway variations have been shown in nature to complete the synthesis of PHE and TYR (16) . In plant chloroplasts, the post -prephenate pathway begins with the transamination of PPA to yield L-arogenate by prephenate aminotransferase (PAT, enzyme 14) . Finally, AGN is either converted to PHE by arogenate dehydratase (ADT, enzyme 15) or to TYR by arogenate dehydrogenase (ADH, enzyme 16) . This pathway is illustrated with dashed arrows in Fig. 1. The basic hypothesis underlying my studies is that a second pathway for aromatic amino acid biosynthesis exists in the cytosol compartment of higher plants. The supportive evidence is that DAHP synthase (the early-pathway enzyme) 5 (32), CM (the mid-pathway enzyme) (23) and ANS (the initial TRP-branch enzyme) (15) are present as pairs of separately compartmented isoenzymes (Fig. 1-2) . It is also known that starting substrates for aromatic biosynthesis (E4P and PEP) are synthesized by duplicate pathways of carbohydrate metabolism located in both the plastid and cytosolic compartments (Fig. 1-2) . Since most secondary metabolites are synthesized in the cytosol of plant cells (39) (Fig. 1- 2) , it seems likely that an aromatic pathway should be available in the cytosol to produce aromatic amino acids not only for protein synthesis but as starting substrates for secondary metabolism as well. Cytosolic isoenzymes of DAHP synthase and chorismate mutase (Ds-Co and CM- II, respectively) are markedly different from Ds-Mn and CM- I (the corresponding plastid- localized isoenzymes) in both catalytic and regulatory properties (32, 23) . Such differences and indeed a lack of homology would be consistent with the endosymbiotic hypothesis of organelle evolution (55) . If cytosolic species of other pathway steps are present in plant cells, it would not be surprising to find them to have equally divergent properties that reflect their xenologous origin at the time of endosymbiosis . Thus, the alternative post- prephenate steps shown in Fig. 1 (solid arrows) might be used in the cytosol. From this perspective it seems unlikely that dehydroquinase and shikimate dehydrogenase CO 0) •H Xi tli V o CQ m •"1 .,-1 ^6 5 2 Xi xi ttCD H [Q o 4-) a o 0) u 4J a at! CQ O •rH to CQ o Eh a m o •H e m u -H 4-) (0 e o x: 4-) 4-1 o o ■H 4-) to 4-) e 4-> O 01 4J x; . nJ 4j a 04 O (0 H m rH ij o CQ a -< -5 -y o (D o ns 4-) ~ CQ O CQ a) -H g u d ns o e u (U tl n3 S o (0 ^ "5 JJ c . >i CQ Xi (fl 0) CN ■H (U CQ O 4-) 0) Xi 4J Tl iH 03 Xi u e 4J M (0 a 0) u B CQ O 0) U ^" N rH C O QJ CQ O o 4-) u o CQ o 4-1 _ >^Xi U CQ O CQ O 4J >i u x: 4-) CQ -H U o5 CQ *^ en i a ^ tn (u ^ > O 0) -H U XI 01 8 would co-exist as a bifunctional protein, as they do in plastids. Alternative possibilities would be that they are monofunctional species, or that they are domains of a large pentafunctional protein like that present in fungi (21) , That the enzymes catalyzing the third and fourth steps in the common pathway of plastids are bifunctional in higher plants has been documented (14, 61) , In £. coli, DQT and SDH are each found as monofunctional proteins (18,19). Yeast and fungi produce the arom protein (21) , a pentafunctional protein bearing functional domains corresponding to steps 2 through 6 of aromatic amino acid biosynthesis (Fig. 1-1) . it is interesting that when the arom pentafunctional protein of Neurospora crassa is subjected to limited proteolysis, the DQT/SDH domains were retained as an intact and functional fragment (77) , Rationale for Focus upon DOT and SDH The rationale for choice of the DQT/SDH protein (or S- protein) for study was as follows, (i) SDH is easily assayed, has very high activity and the substrates are readily available. (ii) The S-protein is in the middle of the common portion of the pathway and provides a link from the early-pathway isoenzyme pair of DAHP synthase to the second known mid-pathway pair of isoenzymes of chorismate mutase. (iii) Two separable shikimate dehydrogenases had been demonstrated by Rothe et al . (67) in pea seedlings, and 9 my preliminary experiments had shown this result to be repeatable in N. silvestris. (iv) Distinctly different properties of the isoenzyme pairs for DAHP synthase, chorismate mutase, and anthranilate synthase have been most useful to demonstrate the separate spatial locations. Since DQT and SDH were known to coexist as a plast id- localized bifunctional protein, I thought it likely that the corresponding cytosolic enzymes would be monofunctional . If so, this would be a type of differential property that might allow me to distinguish the two during cell fractionation studies . Shikimate biosynthesis and guinate catabolism In monocots, such as Zea mays, there are two dehydroquinases, one is bifunctional with SDH (specific for NADP^) and the other is either bifunctional or stably complexed with a NAD'' specific quinate dehydrogenase (36) . The pathway steps to quinic acid in plants have not yet been rigorously established and may originate from E4P and PEP via DHQ, or from unknown pathways. Quinate has been shown to accumulate in plant vacuoles and to account for at least 10%, if not more, of the dry weight of certain plant tissues (2, V. I. Ossipov, personal communications). The quinate catabolic system from fungi (34,35) overlaps the biosynthetic shikimate pathway with several analogous dehydrogenase and dehydratase reactions (Fig. 1-3) . An u 5^ tr W-^" ra rH 5 .C d b i biosynthesis. A al arrows . The ith NAD"^ specific imate dehydratase NADVQDH which al M g J-" s ^H ' s 7^ d ^^ jj ij CO s^ ^ -H g Q a ■H 0 uV ''^ ^ r. ^ ^ .. g ^ nj >< (C i-H m > „, j:^ d -H O 9i 0) -H ate catabol esis is sh shown by th roquinate d The rema Quin; Dsynth em is dehyd acid. . .H 4J - u 7-Q»^^ S M 0) 0 d T3 0^ j-*;^ ^ >,>, --^"S'g ■^ dXi ^ "-d o-H ns 0 0) . -H o 0) QJ 0^ g U -^ T3 tJl U -H tJl 4.) -H "5 rH 0 nJ E- 0 t) ns ^ ,4J • a AJ d ■r^4J^0«a)a)^^>^-H gnsa)MK4J.da)0P nsu'dttc/5m4-i>4-itJ^ u H H o cn < Z Q S H H o s o 11 o X r / / X X i X o z / f^ t M 0> O 2 z n O H K ^ « q o "^ 2 H J u D n n 12 inducible catabolic dehydroquinase takes DHQ that comes directly from quinic acid (by a specific NAD* quinate dehydrogenase) to dehydroshikimate which is converted to PCA and on to carbon building blocks generated by the TCA cycle. The dehydroquinase functional domain of the pentafunctional arom protein of yeast and fungi channels DHQ formed from DAHP to EPSP, thus bypassing PCA formation and avoiding futile cycling (17, 21, 28) . Both a NADPH specific QDH (V. I. Ossipov, personal communication) and a quinate dehydratase (V.I. Ossipov, personal communication) have recently been shown. The NAD"^ specific QDH from E. nidulans also will utilize SHK (34) . These two systems offer some interesting possibilities for overlapping metabolism in plants. For example, it is conceivable that a QDT might convert quinate to SHK for synthesis of aromatic amino acids (Fig. 1-3) to be utilized as precursors for the synthesis of lignin and other compounds. Multiple Species of Shikimate Dehydrogenase Shikimate dehydrogenase has been a popular enzyme choice for a variety of isoenzyme studies and multiple activities have been described on many occasions. The bifunctional DQT/SDH protein (NADP* specific) was examined in dicots (50) . No evidence of a separate DQT or SDH has been determined, thus far. 13 Cofactor specificity. Rot he et al . (67) separated two SDH (NADP*) activities from pea seedlings on a Celite 545 column with an ammonium sulfate gradient. Whether DQT activity coeluted with one or both of the SDH activities was not examined. Shikimate dehydrogenase was shown to be located in the stroma of spinach chloroplasts and was partially purified (30). Mousdale et al . (61) purified a bifunctional DQT/SDH (NADP* specific) from pea seedling chloroplasts and also showed by chromatofocusing that there were two chloroplast isoenzymes and one unstable isoenzyme, probably located in the cytosol . Two forms of nondissoc- iable DQT/SDH (NADP") activities and one of QDH (NAD^) were found in Phaseolus mungo seedlings by Minamikawa (58) . Koshiba (49) also found two forms of DQT/SDH (NADP*) activities from Phaseolus mungo seedlings. In 1988, Ogawa and Tateoka (64) reported one SDH (NADP*) specific activity and two SDH (NAD*) specific activities (DQT apparently was not assayed in these fractions) from Phaseolus mungo. This is the first description of NAD* specific SDH activities and is surprising since the same methodology for separation was used by the three groups and each checked for specificity of cof actors. A recent study of QDH in Phaseolus mungo by Kang and Scheibe (44) found that the enzyme could use NADP* as well as NAD*. When SHK was added to the reaction mix containing quinic acid and NAD*, some inhibition was found. Apparently SHK was not tried as a substrate for QDH. If QDH 1^ 'in' 14 also functions as a SDH, competition for two products (dehydroquinate and dehydroshikimate) would occur and this could account for the inhibition seen with SHK added to the reaction mix. This might also explain the earlier report of the NAD* specific SDH activity by Ogawa and Tateoka (64) , if the enzyme was actually a nonspecific QDH. The qa gene cluster of Neurospora crassa encodes a functional domain for a QDH (NADH specific) that will also utilize SHK/NADH (34) . Properties. The range of molecular mass for DQT/SDH (or SDH, where DQT was not studied) is from 44,000 to 73,000. It is not clear as to whether the lower M^ of 44,000 might represent a monofunctional SDH. SDH activity is usually assayed in the reverse of the physiological direction with the substrates, SHK and NADP*, rather than the forward direction with DHS and NADPH: NADPH Dehydroshikimate < ^ . > Shikiraate NADP" Values of Km have been determined and vary from 0.060 to 1.3 mM for SHK, from 0.010 to 0.10 for NADP% and from 0.130 to 0.180 mM DHQ. Table 1-1 gives estimates of M,. and Km values for DQT and SDH from several plant species, as well as M^ from E. coli and from S. cerevisiae. Inhibition studies with HgClj, p-chloromercuri benzoate and oxidized glutathione have suggested that SDH 15 Table 1-1. Molecular mass and Km values of DQT and SDH, Source Substrate Km imM) M/ Reference Sweet potato SHK 1.300 -- 48 NADP 0.100 Mung bean 1 DHQ SHK NADP 0.130 0.220 0.025 57,000 (GE) 49 Mung bean 2 DHQ SHK NADP 0.180 0.910 0.025 57,000 (GE) 49 Tomato SHK 0.038 73,000 (GF) 52 Pea SHK NADP 0.690 0.013 59,000 61 Corn 1 -- 55,000 (GE) 13 Corn 2 -- 49,000 (GE) Spinach — 67,000 59,000 (GF) (GE) 30 Cucumber SHK NADP 0.060 0.010 44,000 (GF) 53 Radish SHK NADP 0.860 0.090 60,000 (GF) 71 Spinach SHK 0.480 44,000 (GF) 71 NADP 0.032 49,000 (GE) E. coli -- 56,000 (AA) 28'' S. cerevisiae "■ ~ 68,000 (AA) 77 ^Molecular mass estimated by gel filtration (GF) , by PAGE (GE) or calculated from the deduced amino acid sequence (AA) . ''Molecular mass of monofunctional DQT and SDH were combined. sulfhydryl groups act as functional groups in the catalysis of the enzyme and that partial reversal of inhibition is accomplished by thiols (48, 53). Aromatic compounds, metals, and salts have been studied with respect to effect on DQT and SDH activities. Protocatechuic acid was found to be a competitive inhibitor of SDH activity in tomato (52,) and Cl' inhibited DQT activity competitively in pea (61) . Ca^* stimulated SDH activity in Brassica rapa (71) . 16 Molecular genetics. Plant genes encoding DQT or SDH have not been sequenced, but the monocistronic aroD and aroE genes have been cloned and sequenced in E. coli (1, 27) . Amino acid sequences are also available for discrete domains encoding these activities in S. cerevisiae (28) and Aspergillus nidulans (now known as Emericella nidulans) (17) as part of the pentafunctional arom gene iaroM) . The E. coli amino acid sequences, aroD (DQT) and aroE (SDH) exhibit only- have about 25 and 21% identity, respectively, with the corresponding functional domains of the yeast arom protein. In higher plants, aromatic -pathway genes thus far cloned and sequenced are limited to DAHP synthase (29) , shikimate kinase (70), EPSP synthase (73), and chorismate synthase (69) of the common portion of the pathway. Anthranilate synthase (63) and tryptophan synthase jS subunit (3) of the tryptophan branch of the pathway have been cloned and sequenced. The Post-Prephenate Pathway in Plants The dual -pathway hypothesis of aromatic amino acid biosynthesis specifies that an intact post-prephenate pathway exists in the cytosol in addition to that already demonstrated in the plastid compartment (41) . If a separate cytosolic pathway functions: (i) separate genes encoding isoenzyme counterparts of the plastid- localized enzymes may exist, (ii) separate genes encoding alternative enzymes 17 Steps may exist, or (iii) the unprocessed preprotein precursor of the plastid enzymes may function in the cytosol . Prephenate aminotransferase. PAT of higher plants was first characterized in partially purified extracts, separated from aromatic aminotransferases, and shown to be unusually specific for its substrate, prephenate. Activity was optimal at VO^C for PAT, a temperature which inactivated other interfering aminotransferases (5) . PAT was purified about 1000-fold (7) , and localization studies showed the enzyme to be located in the chloroplast (Bonner and Jensen, unpublished data, 1985; 41, 76). A simple, inexpensive spectrophotometric assay was developed for PAT by following the increase in oxaloacetate when the L-aspartate/a- ketoglutarate couple was used (6) . In the same study, the ASP/aKG couple was used with native PAGE activity stained gels to relate the position of PAT with respect to other aminotransferases and to show the disappearance of heat- inactivated aminotransferases. Aroqenate dehydrogenase . Partially purified ADH was studied in N. silvestris with respect to allosteric regulation by TYR by Gaines et al . (31) . ADH was partially purified and regulatory properties were determined in Sorghum bicolor (22) . The enzyme was purified about 1000- fold (8) . PAT, ADH and SDH activities were followed throughout a growth cycle in N. silvestris suspension cells 18 (12) and after wounding of potato (59) . Aroqenate dehydratase . ADT activity was first determined in N. silvestris cells by Jung et al . (43) . The enzyme was localized in spinach chloroplasts and allosteric regulation (activation by TYR and inhibition by PHE) was studied (43) . Siehl and Conn (75) partially purified ADT from Sorghum bicolor and studied some kinetic and regulatory properties. Rationale for focus upon the post-prephenate enzymes. The rationale in choosing to study some aspects of the post- prephenate enzymes for this dissertation is my long-term objective to follow up earlier work with molecular-genetic approaches. Studies of the individual enzymes may shed some light as to the multiplicity of compartment at ion. The merits are as follows. A) Prephenate aminotransferase (i) has been characterized and a well -developed HPLC assay exists (5) , (ii) has important biotechnological application due to its unique aminotransferase properties and the value of its product, L-arogenate, and (iii) can be purified by an established procedure (7) . B) Arogenate dehydrogenase (i) has an established purification methodology (8) for providing purified protein for antibody production that may be used to probe cDNA libraries, and (ii) is easily assayed f luorometrically. C) Arogenate dehydratase has an established HPLC assay (43) , but is by far the most difficult of the three enzymes to assay and purify. 19 CHAPTER II MATERIALS AND METHODS Organisms and extract preparations Eukaryotes Nicotiana silvestris Speg. et Comes suspension cultured cells originally isolated from haploid leaf material have been maintained in our laboratory for about twelve years and are well described physiologically and biochemically (10, 11, 12) . Cell populations are subcultured in stationary phase (7 day) , diluted fivefold into fresh Murashige and Skoog medium (62) , and grown under specified conditions in controlled growth chambers (10) . For these experiments (unless otherwise stated) seven-day old cells were harvested by filtering through Miracloth (Calbiochem, LaJolla, CA) , rinsed with nanopure water, ground in liquid nitrogen in a Waring blender and stored at -70°C. until use. C. sorokiniana cells cultured as described by Meridith and Schmidt (57) were kindly provided by Dr. Robert Schmidt. Bacterial strains and plasmids E. coli strains used for transformation include DH5a, AB1360 aroD (DQT") and SK494 aroE (SDH) . Plasmids used for subcloning were pUC18, pGEMBZf (+) or pBluescript SK+ . 20 Preparation of N. silvestris extract Crude extracts for assay. Frozen, ground N. silvestris cells were suspended (1:1) in 100 mM KPO4 buffer at pH 7.3 containing 20% glycerol, 1.0 mW phenylmethylsulfonyl fluoride and 1.0 mM dithiothreitol, centrifuged at 35,000 x g at 4°C for 20 min. Extracts were treated to 80% of saturation with ammonium sulfate, centrifuged as above, followed by resuspension of pellets in EPPS-KOH buffer (pH 8.6) with all of the above components and dialyzed with four changes of buffer until ammonia could not be detected by HPLC. Extracts not treated with ammonium sulfate were desalted using PDIO columns in Epps-KOH buffer as above or dialyzed overnight with 4 changes of the Epps-KOH buffer. Extracts for purification of ADH and PAT. Five-day N. silvestris suspension cells were used in the purification of ADH and PAT. Crude extracts were prepared as described above and also included 0.1 mAT pyridoxal 5' phosphate and 1.0 mAf EDTA in order to stabilize PAT (5) . Extracts for S-protein purification. For purification of the S-proteins, frozen, ground seven-day N. silvestris suspension cells (300 g) were suspended into 300 ml of 100 mM KPO4 buffer, pH 7.8, containing 20% glycerol, 1 . 0 mM DTT, 1.0 mM benzamidine, 0 . 2 mM PMSF and 0.05 mM leupeptin. A sample was immediately desalted using a PDIO column. 21 equilibrated with 50 mAf EPPS-KOH buffer at pH 8,6, containing the same components as above and labeled as crude extract . Preparation of cultured cells for assay of ADH levels The isogenic suspension cell -culture line of N. silvestris was grown in continuous light with gentle shaking at 150 rev/min. The growth properties of a N. silvestris suspension cell line (ANS-1) in lag, exponential, and stationary phases of growth have been characterized throughout a conventional growth cycle (referred to as E cells) and for cells which have been maintained in exponential growth for more than 10 generations, EE cells (10) . For routine subculture, 80 ml of suspension-culture medium were inoculated with 20 ml of 7 -day stationary-phase cells in 500-ml Erlenmeyer flasks. EE cells (2 g wet weight) were transferred to fresh medium on day 7 when cells reached about 1 X 10^ cells ml"^. Daily samples were harvested using Miracloth for filtration, washed with distilled water, ground and frozen in liquid nitrogen, and stored at -70°C until extract preparation. Daily sampling was also done to determine a cell count by mixing 1 ml of a 50 ml culture with 12% chromium trioxide (helps to separate cell clumps and visualize cells more easily) . Samples were heated to 70°C for 2 to 5 min before counting in a Fuchs- Rosenthal counting chamber at 20x magnification. The remaining 49 ml of culture were filtered and cells were ■:-M .OSJ ;^ 22 dried on preweighed filters made from Miracloth in an oven at 50°C and were weighed daily until constant dry weight values were obtained. From these two methods, growth curves were determined. Preparation of C. sorokiniana extracts Frozen cell pellets of C. sorokiniana were suspended into 50 mM PIPES buffer, pH 7.3, containing 1.0 mAf DTT, 1.0 mAf PMSF, 35 mM KCl , and were broken by 2 passes through a French Pressure Cell at 20,000 psi. The slurry was centrifuged at 18,000 x g, at 4°C for 15 min, and the supernatant was next centrifuged at 150,000 x g for 1 h at 4°C. Extracts were either desalted and used for column chromatography, or dialyzed with 50 mM EPPS-KOH buffer with all protective components as listed above for use in enzyme assays. (Pipes buffer plus KCl was used in place of EPPS buffer for column chromatography in order to separate isoenzymes of chorismate mutase for another study.) Glycerol and PLP were added to column fractions to protect several of the enzymes to be assayed (PAT and ADH) . Preparation of bacterial extracts The mutant E. coli strains {aroD and aroE) carrying the cDNA clones encoding functional proteins (DQT or SDH, respectively) were grown in 200 ml of LB/Amp media, washed and resuspended in KPO4 buffer containing 2 0% glycerol and 1 mM DTT at pH 7.6, and centrifuged at 150,000 x g, at 4°C for 23 1 h. The supernatant s were desalted by passage through Sephadex G-25 columns before fluorometric assay. Enzyme assays Aminotransferases Prephenate aminotransferase. Prephenate aminotransferase was usually incubated with concentrations of 20 xnM L-glutamate and 0.8 mW prephenate (unless otherwise stated) , incubated at 37°C for 10 min (or as otherwise stated) and then assayed by HPLC as an orthopthalaldyhyde derivative in a 60% methanol/40% of 20 mM KPO4 buffer system. Peaks of PHE were quantified before and after acidification of the product of the reaction, AGN, in IN HCl for 10 min at 37°C. Aminotransferase couples. Other aminotransferase assays included an amino acid donor (ASP, TYR, LEU or ALA) and keto acid (PPY) at concentrations as stated and were assayed similarly by HPLC, before and after acidification when applicable. Controls included samples without amino acid donor or keto acid. Aroaenate dehydratase Arogenate dehydratase was assayed according to Jung et al. (43) by measurement of OPA derivatives following HPLC where the peak of AGN formed were monitored before and after acidification to PHE. Concentration of 0 . 5 mM AGN, 0.25 mAf TYR (activator), 50 mM EPPS-KOH buffer at pH 8.6 and enzyme .J 24 were included in the reaction sample of 100 /xl that were incubated at 32°C for 3 0 min. Controls included samples without AGN or enzyme. Dehydroquinase Dehydroquinase activity was assayed spectro- photometrically at 234 nm at room temperature (24°C) by following the rate of increase of product, dehydroshikimate, when dehydroquinate was provided as substrate . Unless otherwise stated, a reaction sample included 0.5 mAT DHQ, enzyme and 50 mM EPPS-KOH buffer at pH 8 . 6 . An extinction coefficient of 12,000 was used to calculate activity (37). DQT was also assayed f luorometrically via a coupled assay with SDH. DHS formed from DHQ by the dehydratase reaction was reduced with NADPH (cof actor for SDH) and the rate of NADPH disappearance was followed. Reaction samples contained 0 . 5 mM DHQ, 0 . 2 mAf NADPH in 50 mM EPPS-KOH at pH 8.6 for column fractions or 50 mM Bis-tris propane buffer, pH 7.5, for other studies as stated. Controls were carried out for each enzyme assayed by adding only one substrate for about three to five min before adding the second substrate. Dehydrogenases Dehydrogenase reactions were usually assayed fluorometrically at room temperature (24°C) with cofactor NAD(P)* for 1 to 2 min before addition of substrate, and the rate of formation of NAD(P)H was monitored (340 nm emission 25 and 460 nm excitation) . Unless otherwise stated, a total vol of 400 /xl contained substrates and 50 mM EPPS-KOH buffer at pH 8.6. The rate is expressed as f luorometric units per min (FU min-^ . Activities were calculated as nmol min"^ by relating experimental values to a standard curve obtained with authentic NADPH. Any background rate seen when cofactor was present alone in reaction sample (only in some crude extracts) was subtracted from the rate obtained from reaction sample containing both substrates to give an appropriate correction. A spectrophotometric assay was also used occasionally by following the rate of formation of NAD(P)H at A340 nm- Aroaenate dehydrogenase . Arogenate dehydrogenase activity was determined when 0 . 5 mM AGN was added to the reaction assay after 0 . 5 mAT NADP* had been added or as otherwise stated. When appropriate, NAD* was added as cofactor to the reaction mix. Prephenate dehydrogenase . Prephenate dehydrogenase activity was assayed with 0.5 mAf NADP* (or NAD*) and 1 . 0 mAT PPA. Shikimate dehydrogenase . Shikimate dehydrogenase activity was usually assayed in the reverse-of -physiological direction with 1 . 0 mM NADP* and 4 . 0 mM SHK, unless otherwise stated. When appropriate, NAD* was added to the reaction sample. In the forward direction, SDH was assayed with 26 concentrations of 1.0 mM NADPH and 1.0 mM DHS or at concentrations otherwise stated. Ouinate dehydrogenase . Quinate dehydrogenase was assayed with 1.0 mM quinate and 1.0 mAf NAD* or NADP* in 100 mM glycine buffer at pH 9.5. Prephenate dehydratase Prephenate dehydratase was assayed by incubation of PPA and enzyme in 50 mM EPPS buffer (total vol of 100 ^1) at 37°C for 10 to 20 min before addition of 100 fil IN HCl followed by further incubation at 37°C for 10 min. Controls included PPA or enzyme with buffer only. Activity was calculated from spectrophotometric readings at A320 nm arid an extinction coefficient of 17,500. Protein estimations Protein concentrations were estimated by the method of Bradford (14) with BSA as a standard. A micro-assay was used, as described, to estimate highly purified protein concentrations . Definition of activity and specific activity Activity for all enzyme reactions in this study are defined as nmol min"^ for the stated amount of protein added into a standard assay volume. Specific activity is defined as nmol min'^ mg protein'^. 27 Separation of pathway enzymes by chromatography Column chromatography of N. silvestris extracts A DEAE- cellulose column for separation of enzymes of aromatic biosynthesis was prepared similarly as described under purification. A 100 mg amount of protein was applied to a bed volume of 102 ml of a DEAE column equilibrated in 50 mAT KPO4 at pH 7.5. After washing the column with about 3 column volumes of buffer, a 600 ml gradient from 0 to 0.4 M KCl (KPO4 buffer) was applied. Column chromatography of C. sorokiniana extracts A DEAE-cellulose column (20 ml) was prepared, equilibrated in Pipes buffer (above) , and about 29 mg of protein from Chlorella were applied. The column was washed with about 40 ml of the same buffer before a 35 mM to 500 mM KCl gradient was added. Finally, 1 . 0 AT KCl/Pipes buffer was added to remove residual protein from the column. Appropriate fractions with DQT and SDH activities in the gradient were pooled and dialyzed against 5 mAf KPO4 buffer, 20% glycerol, and 1 mM DTT at pH 7 . 2 . An HA column (10 ml) was prepared with the 5 mM KPO4 buffer, pH 7 . 2 . A 15 -ml sample from above was applied to the HA column and activities for DQT and SDH eluted in the wash fractions. Celite 545 (shallow-gradient application of Fig. 5-1) A 200-g amount of ground, frozen 7-day suspension cells was suspended in 200 ml of KPO4 buffer, 7.2 pH, containing protectants and centrifuged as described above. A 3 -ml 28 amount was removed for dialysis in Epps-KOH buffer and used as a crude extract. Ammonium sulfate was added to give 75% of saturation. From the slurry, 70 ml were removed and prepared for use as a concentrated crude extract . To the remaining slurry, 40 g of Celite 545 was added, stirred, poured into a column (90 ml vol) and the eluate collected into a cylinder. The column was washed with 65% (NH4)2S04 of saturation in Epps-KOH buffer (about 180 ml) and 8-ml volumes were collected. Buffer with (NH4)2S04 at 55% of saturation (120 ml) was added, followed by a shallow gradient of 600 ml from 55% to 45% of (NH4)2S04 saturation in Epps-KOH buffer. A step gradient of 180 ml of buffer at 45% of (1^4)2804 saturation was applied before the second gradient of 45% to 0 of (NH4)2S04 saturation (400 ml) was added. A final wash of 120 ml of Epps-KOH buffer completed the elution schedule. Gel filtration A Sephadex G-200 gel filtration column was used to determine the native molecular weight of the S-proteins. The column was equilibrated with 50 mM Epps buffer, pH 8 . 6 and DTT. Three ml of concentrated crude extract, and concentrated SP-I or SP-II combined with 0 . 1 Af sucrose were applied individually to the same column (column vol of 471 ml, void vol of 119.4 ml) . Protein standards of 5 mg each. 29 alcohol dehydrogenase (150,000 D) , BSA (66,000 D) and carbonic anhydrase (29,000 D) plus sucrose were applied to the column. Purification of pathway proteins Purification of the S-proteins Crude extract of 54 0 ml was brought to 60% of ammonium sulfate saturation at 4°C while stirring and was used for the purification of the S-proteins. Celite 545 chromatography. After stirring the 60% of ammonium sulfate saturation slurry for 3 0 min, 70 g of Celite 545 was added, with continued stirring for about 20 min. The slurry containing 605 mg protein was applied to a column and the eluate collected into a large cylinder. The column volume of 174 ml was washed with 60% of (NH4)2S04 saturation-Epps-KOH buffer containing 20% glycerol and 1.0 mM DTT at pH 8.6 and was collected in a separate cylinder. No activity was found in these eluates, although the protein level was very high. A 500-ml gradient from 60 to 40% of (NH4)2S04 saturation in the same buffer was applied and 8-ml fractions were collected. Next a 40% of (NH4)2S04 saturation-Epps-KOH buffer of about 130 ml was applied. A second 400 ml gradient of 40 to 0% of (NH4)2S04 saturation- Epps-KOH buffer was applied, followed by a wash of buffer without (NH4)2S04. Two peaks of SDH/DQT activities were located, pooled individually, and dialyzed against the Epps 30 buffer as above. Samples of each were saved for assay, and the remainder of each pool was used in the second purification step (643 ml, 206 mg of SP-I protein and 348 ml, 97 mg of SP-II protein) . CM-cellulose chromatography. CM cation exchanger columns were equilibrated with 50 mM Epps-KOH, 20% glycerol and 1 mM DTT at pH 8.6. SP-I was applied to a bed volume of 123 ml and SP-II was applied to a bed volume of 80 ml. Both SP-I and SP-II eluted in wash fractions and a small portion was saved for assay. The remaining portions of the preparations were used in the third step of purification. DEAE-cellulose-52 chromatography. Two DEAE-52 anion exchanger columns were prepared and equilibrated in the same Epps-KOH buffer as the preceding step. Proteins were applied, SP-I to bed volume of 107 ml and SP-II to a bed volume of 72 ml, and washed with the same buffer. A 4 00 -ml (SP-I) or 300-ml (SP-II) gradient from 0 to 0.6 mM KCl was applied to the columns. Fractions of about 6 . 5 ml were collected. Single peaks of activity, each eluting at about 0.3 mAf KCL were individually pooled and dialyzed against 5 mM KPO4 buffer containing 20% glycerol and 1 mM DTT at pH 7.3. Samples were saved for assay, and the remainder of each protein was used in the fourth step of purification. Hydroxylapatite column chromatography. SP-I and SP-II were applied to HA columns equilibrated in the KPO4 buffer of the previous step (100 ml and 50 ml bed volumes. 31 respectively) . Each protein eluted in the wash and was applied to the fifth step of purification after samples were removed for assay. DEAE KPO^ chromatography. DEAE columns were equilibrated in 50 mM KPO4 buffer at pH 7.3, before applying the proteins from the previous step. Gradients up to 0.3 M KCl were applied to each column. Single peaks of each protein were eluted, pooled and dialyzed into 50 mM Epps-KOH containing 20% glycerol and 1 vnM DTT at pH 8.6. Samples were saved, and the remaining proteins were subjected to the sixth step of purification. 2'5'ADP Sepharose-4B affinity chromatography. An NADP*- specific 2'5'ADP Sepharose-4B affinity column was prepared and equilibrated with the Epps-KOH buffer. The SP-I sample was applied to the column and washed with starting buffer before a 300-ml gradient from 0 to 0.18 M NaCl and 0 to 12 @8,25 fiM NADP* was applied. The protein eluted at about 4 fiM NADP* in the gradient. The SP-I Activity peak was pooled and dialyzed into the Epps-KOH buffer. A sample of SP-I was saved, and the remaining fraction was used in the seventh step of purification. The SP-II was applied to the affinity column similarly. Purification of SP-II was completed with this step at a final volume of 15 ml and total protein of 6 ng . 32 Final steps of SP-I purification. SP-I was applied once again to a DEAE column equilibrated in Epps-KOH buffer as previously described. A 200-ml gradient from 0 to 0.6 Af KCl was applied. The SP-I activity peak fractions were pooled and dialyzed against Epps buffer. An aliquot was saved, and 83 ml containing 0.162 mg protein was applied to the affinity column for the final step of purification. The completed purification yielded 0.07 mg SP-I protein in 54 ml. Purification of arogenate dehydrogenase ADH was purified by two methods: A) by a series of steps of an established procedure which began with a 0 to 45% ammonium sulfate fractionation and ended with the NADP* affinity column (8) , or B) by the elution of ADH from a Celite 545 column used to separate S-protein activities, followed by HA column chromatography, DEAE column chromatography, and the NADP* affinity column chromatography described above . Purification of prephenate aminotransferase PAT was purified by following an established procedure (7) , which began with a 45 to 75% of ammonium sulfate saturation precipitation step, followed by a 70°C treatment to inactivate and then precipitate (by centrifugation) contaminating aminotransferases, and ended with a specific aminotransferase pyridoxamine phosphate (PMP) affinity column . 33 PAGE SDS gel electrophoresis SDS gel electrophoresis was prepared by following the procedure of Laemmli (51) , with a 15% running gel and a 4% stacking gel . A running buffer of Tris/glycine/SDS at pH 8.8 was used and the gel was run at constant voltage of 63 V for about 8 h. The gel was stained for about 3 0 min in Coomassie Brilliant Blue R250 dye, destained and viewed. Silver staining Silver staining of an SDS gel as described by Pharmacia (Piscataway, NJ) was followed. Washes of methanol/acetic acid and ethanol/acetic acid were included before incubation of the gel in oxidizer, followed by addition of the silver stain, then developer, and finally an acetic acid/water wash to stop the reaction. Native gel electrophoresis A native PAGE was run according to the method of Shaw and Prasad (74) . Gel concentrations were 7% for the stacking gel and 15% for the running gel. The running gel was pre-electrophoresed to remove the ammonium persulfate, and fluorescent light was used to solidify the stacking gel which contained riboflavin. The gel was run at constant voltage of 60 V for about 8 h. Shikimate dehydrogenase activity stained gel Optimal conditions for SDH activity stain on native PAGE included 20 mg NADP\ 50 mg shikimate, 10 mg 3- (4,5- 34 dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium-bromide; (thiazolyl blue) , and 2 mg N-methyldibenzopyrazine methyl sulfate (phenazine metasulfate) in 50 ml of 100 mAT glycine '] "■i buffer (without glycerol) at pH 9. Gels were incubated at 37°C for 10 to 20 min or at room temperature for 30 min to an hour. These conditions were used unless otherwise stated and were modified from the method presented by Davis (24) , Kinetic values Shikimate dehydrogenase Enzyme activities were assayed f luorometrically as described for SP-I and SP-II. In the forward physiological direction, either DHS or NADPH was held constant at saturating levels while the remaining substrate concentration was varied to obtain saturation curves. In the reverse of physiological direction, SHK and NADP* were assayed similarly. Double reciprocal plots of the substrate saturation data were used to determine the Km for each substrate. Dehydroquinase DQT activities were assayed spectrophotometrically as described for SP-I and SP-II at varying concentrations of DHQ to obtain saturation data, and then analyzed appropriately as above to determine the Km for each protein. ,1 i 35 Inhibition studies of S-proteins Enzyme was incubated for 10 min with p-chloromercuri- benzoate or with possible protectants {DTT, cysteine or fS- mercaptoethanol (jSME) } , SDH was assayed at saturating conditions and DQT was assayed at 0.1 xnM DHQ. For prevention experiments, DTT, cysteine, or jSME was added about 10 min before addition of substrates and PCMB, and then rates were determined. For reversal experiments, PCMB was added to enzyme and buffer for 10 min before assay with substrates. After a rate (or no rate) was established, DTT was added to the reaction mix. Concentrations are as stated in Results. Antibodies Preparation of specific antibodies Antibody preparations were produced by Kel- Farms (Alachua, FL) with repeated injections of purified SP-I over the course of several months until antibody titers, tested by Ouchterlony double diffusion method against SDH, ADH or PAT, were high enough to bleed the rabbits. Purification of specific antibodies Antibodies were purified following a BIO-RAD (Rockville Center, NY) procedure, by passing each over Econo-Pac lODG columns for desalting and then passing through DEAE Affi-Gel Blue Econo-Pac lODG columns for serum IgG purification. E. coli strain DH5a lysate was prepared and incubated with the 36 purified IgG. This step removed non-specific precipitant bands observed in the double-diffusion experiments. Pre- immune rabbit serum were purified similarly. Antigen; antibody assay Antigens (SP-I, ADH or PAT) from crude extracts or extracts from various purification steps were combined 1:1 (v:v) (or as stated) with antibodies. Samples were incubated at 37°C for 10 min and returned to an ice bath, then centrifuged (5 to 10 min) at full speed in a microfuge to precipitate the complexed antigen: antibody. The supernatants were assayed for enzyme activities. Controls were always performed with preimmune rabbit serum. Ouchterlony assay Ouchterlony plates were prepared with 1 . 5% agarose in sodium barbital and sodium azide and wells were prepared at precisely measured distances (66) . Antibodies or antigens were diluted with 0.9% NaCl whenever necessary and then added to the wells. Plates were incubated at 37°C overnight . Western blotting Western blotting procedures from Sambrook et al . (68) were followed, and bands from either SDS or native PAGE were transferred to nitrocellulose filter paper. Membranes were incubated in blocking solution alone and in blocking solution containing diluted SP-I antibody for one h, washed, incubated further with blocking buffer containing alkaline 37 phosphatase -linked goat -anti -rabbit antibody for 1 h, and then incubated in STP buffer containing MgClj and substrates, nitroblue tetrazolium and the p-toluidine salt of 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, membranes were viewed and photographed. Antibody precipitation of DOT and SDH from C. sorokiniana Antibody made to SP-I was incubated 1:1, 2:1, or 4:1 with Chlorella extract (controls included preimmune antiserum and extract diluted with buffer) . After 10 min at 37°C, and centrifugation for 5 min at full speed in a microfuge, preparations were assayed under standard conditions for SDH and DQT activities. cDNA cloning and sequencing Tobacco leaf cDNA library A cDNA library prepared from Nicotiana tabacum var. SRI tissue culture cells, inserted into pBluescript SK- plasmids and packaged into Lambda ZAP II vectors, was purchased from Stratagene, La Jolla, CA. E. coli XLl-Blue (a host strain used for immunological screening of libraries constructed in Lambda ZAP expression vectors) and E. coli SOLR (a non- suppressing host strain for use after excision of the pBluescript plasmid from the Lambda Zap vector) were included. Helper phage, ExAssist, efficient for in vivo excision of the plasmid from the Lambda Zap vector without replication of the phage genome was also included. 38 Preparation of cDNA libraries for screening The cDNA library was first titered, then mixed with the freshly prepared host strain, XLl-Blue, plated with top agar on about 80 LB plates at about 20,000 PFU (about 1.6 X 10® PFU/ desired clone) and incubated at SV^C. Isopropylthio-/8- D-galactoside treated nitrocellulose filters were applied about 6 to 8 h later and incubation was continued overnight. Filters were removed and the immunological screening procedure of Sambrook et al . (68) was followed. Screening the cDNA library of Lambda ZAP II Nitrocellulose filters removed from the plates were washed, soaked in blocking buffer containing dry milk, rocked gently in primary S-protein, ADH or PAT specific antibody/blocking buffer and washed several times. The filters were next exposed to secondary antibody, goat anti- rabbit antiserum, washed as before and incubated with substrates, nitroblue tetrazolium, 5-bromo-4-chloro-3- indolyl phosphate and with 5 mAf MgClz. The filters were kept in the dark and gently rocked for one to two hours. Plaques corresponding to the small, deep blue spots on the filters were cored from plates, dispensed into SM buffer plus chloroform and stored at 4°C for titering and plaque purification. . -,1 39 Excision of Pbluescript from the Lambda ZAP II vector Purified phage stocks were incubated with E. coli strain XLl-Blue and ExAssist helper phage to excise the pBluescript plasmid from the Lambda ZAP II vector for 2 to 2.5 h at 37°C in 2XYT media (see Stratagene instruction manual for details) , heated to 70°C and centrifuged. The supernatant, containing the plasmid, was incubated with host E. coli strain SOLR, spread on LB plates containing 50 mg ml"^ ampicillin and incubated overnight at 3 7°C. Colonies from these plates were streaked on fresh LB-AMP plates for immediate use and were also stored in DMSO for future use. DNA purification Plasmid DNA was purified by two methods. Method 1: The pBluescript plasmids containing putative cDNA clones coding for the S-protein, ADH or PAT were purified for sequencing by the method of the DNA Sequencing Core Facility, ICBR, University of FL. A colony from the E. coli SOLR strain from the LB -AMP plate above was incubated overnight in 10 ml of LB-AMP medium in a 50 ml tube for each clone sample. The cultures were centrifuged and the pellets were resuspended in GTE (68) buffer. Treatment with NaOH/SDS was followed by neutralization with potassium acetate at pH 4.0, and centrifugation was carried out to remove cellular debris. The supernatant s were treated with DNase-free RNase and extracted twice with chloroform. DNA was precipitated with isopropanol and washed with 70% ethanol . To precipitate the 40 plasmid DNA, the pellets were first dissolved in sterile water, then 4M NaCl and 13% PEGgooo was added, followed by incubation on ice before centrifugation at 4°C. The pellets were rinsed in ethanol, dried under vacuum and resuspended in sterile water. DNA concentrations were determined spectrophotometrically at 260 nm and by agarose gel electrophoresis with a known DNA standard. Method 2: The 10 ml cultures, as prepared above after centrifugation, were treated with prepared solutions provided by Promega (Madison, WI) in the Magic Miniprep DNA purification kit. The DNA was passed through a mini -column and dissolved in GTE (Promega instructions for preparation) buffer. This rapid method is preferred for sequencing on a Li -Cor sequencer available in the Department of Microbiology and Cell Science. Competent cells of E. coli strains E. coli strains DHBo;, AB1360 {aroD) and SK494 [aroE) were grown overnight in 50 -ml tubes and inoculated into 1-L flasks containing 200 ml LB medium. The cells were incubated at 37°C in a shaker at 3 00 rpm for several hours until a spectrophotometric reading at Agog nm between 0.4 and 0.5 was reached. Cells were harvested by centrifugation in sterile tubes after incubation on ice, treated with 100 mM CaClz as described by Sambrook (68) , resuspended into 100 mM CaClj with 15% glycerol, aliquoted into microcentrifuge tubes, dropped into liquid nitrogen and stored at -70°C 41 until use. Controls were prepared by streaking auxotrophic mutant strains on M9 medium plates +/- aromatic amino acids. Transformation of plasmid DNA Either pUC18, pGEM5zf(+), or pBluescript SK+ plasmids were incubated with competent cells of DH5q; on ice, heated to 42°C for 90 sec, cooled in an ice bath before addition of SOC medium (68) . The cells were then incubated at SV^C for 1 h before spreading on LB-AMP plates. A colony from these transformed cells was streaked onto a fresh LB-AMP plate. Controls included spreading of competent cells without plasmid or plasmid without insert onto LB-AMP plates. Functional complementation E. coli mutant strains, aroD (DQT") and aroE (SDH") , were prepared for transformation with the pBluescript plasmids carrying cDNA clones and were plated on LB-AMP plates. After growth, colonies were streaked onto M9 plates. Controls included nontransformed competent cells or plasmid spread on LB-AMP plates, and competent mutant strains spread on M9 plates +/- aromatic amino acids. Initial sequencing of cloned cDNA Purified pBluescript plasmid with cloned inserts were submitted to the DNA Sequencing Core Facility, ICBR, UF, for initial 5' and 3' end sequencing (from the T3 and T7 promoter sites) . 42 Subcloninq cDNA for complete sequence analysis Subcloning of cDNA inserted into pBluescript was performed by use of appropriate restriction enzymes to obtain fragments for sequencing both strands of the entire cDNA. Plasmids used for fragment insertion {pUC18, pGEM5zf(+), or pBluescript SK+} were incubated with the same restriction enzyme (s), and were treated with calf intestinal alkaline phosphatase (CIAP) (when only one RE was used) to prevent rejoining of ends during ligation. Cloned cDNA fragment samples and plasmid samples were loaded onto 1% DNA agarose gels at 70V for about 1 h. Fragments and linear plasmid DNA were excised and treated with the Gene Clean system (Bio 101, LaJolla, CA) , prior to ligation and transformation . Li-Cor sequencincT of subcloned cDNA fragments Plasmid DNA was denatured and reacted with forward or reverse fluorescent primers, enzymes, 7-Deaza dGTP sequence extending mix and NTPS according to the directions supplied by Li-Cor (Lincoln, NE) . Three /il of each of four prepared sample mixtures containing one of the four dideoxy nucleotides was loaded onto wells of a urea polyacrylamide gel set into the Li-Cor model 4000L sequencer. Parameters were set in a computerized data collection file and the sequencer was run overnight . The sequences were analyzed in a data analysis computerized system. 43 N-terminal sequencing About 1 pmol of purified SP-I protein was prepared for N- terminal sequencing on SDS PAGE, followed by transfer of the protein onto 3MM paper by electrophoresis. Mini-blot system protocol and amino acid analysis by acid hydrolysis was provided by the Protein Chemistry Core Facility, Interdisciplinary Center of Biotechnology Research, University of Florida. Computer analysis The GCG computer software package (26) was used to analyze nucleotide and deduced amino acid sequences of cloned cDNAs . Biochemicals Arogenate (90% pure) was isolated from a multiple auxotroph of Neurospora crassa ATCC 36373 (42) or isolated from a tyrosine auxotroph of Salmonella typhimurium (4) . Prephenate (85% pure) was prepared as the barium salt from culture supernatants of the tyrosine auxotroph of S. typhimurium and was converted to the potassium salt with excess K2SO4 prior to use. Dehydroquinate (90% pure) was chemically synthesized by following the method of Haslam et al . (37) using a platinum-catalyzed dehydrogenation of quinic acid (about 90% pure) . 2' 5' ADP Sepharose-4B and AH- Sepharose 4B were purchased from Pharmacia, Piscataway, NJ. Bradford reagent was purchased from Bio-Rad, Rockville 44 Center, NY. DTT and hydroxyl apatite were purchased from Research Organics, Inc. Cleveland, OH. Shikimate, NADP, NADPH, benzamidine, leupeptin, and PMSF were purchased from Sigma Chemical Co., St. Louis, MO. All other chemicals were purchased through Fisher Scientific Co., Orlando, PL. 45 CHAPTER III PHYSIOLOGICAL PROFILE OF AROIVIATIC PATHWAY ENZYMES IN PLANTS The activities of dehydroquinase and shikimate dehydrogenase (of the common portion of aromatic biosynthesis) , and prephenate aminotransferase, arogenate dehydratase and arogenate dehydrogenase (of the post- prephenate portion of the pathway) were examined under selected physiological conditions in the higher plant N. silvestris and in the green alga Chlorella sorokiniana. Prephenate aminotransferase from Chlorella exhibited several of the striking characteristics of higher plant enzyme. Arogenate dehydratase activities were followed in a growth culture cycle of N. silvestris suspension cells similarly as was followed for PAT and ADH (12) . Results Specific activities from crude extracts Specific activities have been compared for several aromatic amino acid pathway enzymes of N. silvestris cultured cells and from the green alga C. sorokiniana (Table 3-1) . A constant ratio of about 5 for SDH and DQT activity was found in N. silvestris, compared to a ratio of 1 for crude extracts of C. sorokiniana. The activities of DQT and 46 SDH from Chlorella extract were unaffected when incubated with SP-I N. silvestris specific antibody. Activity was not found when QDH was assayed in N. silvestris and C. sorokiniana crude extracts. Table 3-1. Specific activities from N. silvestris and C. sorokiniana aromatic pathway enzymes. Specific Activity (nmol min"-"- mg"-"^ protein) Enzyme N. silvestris C. sorokiniana QDT 12.00 6.20 SDH 60.00 6.00 PAT 16.00 3.75 ADH/NADP 1.00 NF ADH/NAD NF NF PDH/NADP NF NF PDH/NAD NF NF ADT 0.6 NF PDT NF NF NF, not found; Crude extracts were used for assay. Separation of activities Column chromatography was used to separate the enzymes of interest for this study from N. silvestris and C. sorokiniana. DQT and SDH of the common pathway were not separable in N. silvestris or in C. sorokiniana after two steps of column chromatography (DE-52 and HA) . Figure 3-1 shows a DEAE-cellulose chromatographic profile of N. silvestris suspension cell extract with overlapping . CO T) ^ 03 4J e c :3 0 g iH u d 0) M-l iH 0 ,.^ CQ u • 0) ^^ IT) m N 1 Q c w w 0) Q TJ >1 ns C OS rt ^ 0 ^ 4J » 4-) T) 'i^ Dj •H ^■^ u rH E-" •H 4J a tt § (D fO ■ 0 0] _J^ '7 jS n 0 CO m Q) rH § Sh 0 >, Q) 4J X! U * -H a ^^ en 0 i-> e 0 C < d 0 0 -H to {-< B CO C < m 0) CO CQ CQ 73 -H (U 4J o u >i 0 0) to OS Xi (U 0 3 T3 rH 4H C nH nS 0) IW 1 u 1 0 CQ rH 1 s (0 -H 4J [l] to u c Q M 0) OJ 4J 4J ■H X nJ T) • w s nS H ^ 1 C en ro • •H CQ ,-{ . •H T) U tj) u 0) t>d -H -U XI b CQ •H 0) ^ u x: > u 4-» M CQ •H 0) c CQ T> ■rl w ■^ q (O CVi 00 "* CNi CVJ *- ■»- o o 1 1 1 1 1 1 in ^ C3 CVi f- o o o o o 48 V- ui.LU nj 8 lA Q LO P U) O U) 55 c5 CVi ^ -r- T- OlcJH ( 9 01.x) V3yv 49 enzyme activities of PAT, ADH, ADT and SDH (DQT activity was coincident with SDH activity; not shown) . Although ADT and ADH activities were not found in crude extracts of C. sorokiniana, they were located in DEAE-cellulose fractions at very low activities as shown in Fig. 3-2A. ADH was specific for cof actor NADP*. Prephenate dehydratase and prephenate dehydrogenase activities were not found in these column fractions. The ADH and ADT activities overlapped those of PAT, DQT, and SDH in the KCl gradient fractions. The coeluting DQT and SDH activity peak fractions, as shown in Fig. 3-2B, were pooled, dialyzed and applied to an HA column where they once again coeluted, this time in the wash fraction, data not shown. PAT in C. sorokiniana Several of the unique properties earlier shown for prephenate aminotransferase in N. silvestris suspension cells (5) were studied in C. sorokiniana extracts. A high temperature optimum of about 70°C for PAT was also found in the alga extract as shown in Fig. 3-3. In Table 3-2, it is shown that PAT from Chlorella has a preference for GLU as the amino acid donor in combination with the keto acid substrate, PPA. As found in higher plants, ASP was utilized at about 50% of the activity with GLU. Slight activity was seen with TYR as the amino acid donor in C. sorokiniana but not in N. silvestris . Fig. 3-2. DEAE-cellulose chromatography of aromatic pathway enzymes from C. sorokiniana. Alga extract was applied to a DE-52 column as described in Materials and Methods. A) PAT (•) , ADH (■) , and ADT (a) eluted in the KCl gradient (-) and protein was monitored (...) . B) SDH (O) and DQT (•) eluted in the gradient. 51 to tn < o. X o tn < 1.0 1.8 0.8 1.6 0.8 1^ 0.4 5 OS 0.2 0.4 0 _ 0 s CM 20 40 60 FRACTION NUMBER 80 1.0 1.8 0.8 1.8 0.8 o 0.4 5 0.2 0.4 0 0 20 40 60 FRACTION NUMBER 52 X H H > H H U < 20 40 60 80 TEMPERATURE (°C) Fig. 3-3. Temperature optimum for activity of prephenate aminotransferase in C. sorokiniana. _ The temperatures range used was from 20°C to 80°C for 5 min. 53 Table 3-2. Prephenate aminotransferase amino acid donor specificity in C. sorokiniana. L-Amino Acid Donor (10 mM) Specific Activity GLU leTo ASP 8 . 0 TYR 2 . 0 LEU 0 ALA 0 ^A concentration of 1 mAf PPA was used in combination with the amino acids. S. A. is nmol min" mg protein' When C sorokiniana extract was incubated at 65°C for 10 min and then assayed at 37°C, activity with PPY as keto acid substrate was lost, but activity with PPA remained constant. At 70°C for 20 min, however, about 60% of the PAT activity was lost (Table 3-3) . Table 3-3. Prephenate aminotransferase activity after thermal treatment in C. sorokiniana^. Aminotransferase Couple No treatment 65"C 10 min 70"C 10 min 70"C 20 min PPA/GLU PPY/GLU 0.61 0.29 0.60 0 0.61 0 0.25 0 ^Reaction mixtures were incubated at 37*C following thermal treatment. Assays contained 1 mM PPA or 10 mM PPY, 10 mM GLU and 0.05 mg protein. 54 APT activities in cultured N. silvestris cells. When N. silvestris stationary phase cells were diluted into fresh medium, a substantial elevation of soluble protein occurred which increased until day 2, leveled off between days 2 and 3, and began a steady decline between days 3 and 7 as shown in Fig. 3-4B. This rise ceased before mid- exponential growth was reached. That soluble protein synthesis was terminated before reaching a maximum was indicated by the finding that EE cells achieved a two- fold higher content of soluble protein. EE cells exhibited a consistently higher soluble protein content of about 360 mg g dry wt.'-^. During the transition of cell populations in lag phase to exponential -phase growth, new synthesis of macromolecular constituents must have occurred. Figure 3-5A shows that the rise in ADT activity expressed as units of activity per g dry wt . , paralleled the rise in soluble protein between subculture and day 2 (Fig. 3-4B) . The rise in ADT activity levels continued until day 3, whereas overall soluble protein content plateaued between days 2 and 3. Thus, specific activity or activity expressed on a cell basis peaked at day 3. Since the specific activity of E cells and EE cells are similar and since the ratio of soluble protein to dry weight is greater in EE cells than in E cells, the activity of ADT expressed as units per g dry wt was greater in EE cells than in E cells. The decline in specific Fig. 3-4. Growth curve of N. silvestris in suspension cultures. A) Cell growth monitored as cell count or as dry weight. The physiological stages of growth: lag (L) , exponential (E) and stationary (S) are indicated in each panel. B) Soluble protein content was determined in samples harvested daily and was related to the dry wt . and cell number. Comparable protein values obtained with EE cells (average of ten determinations) are indicated with stippled bars, the widths of which indicate the range of variation. 56 3x10* 2x10*- L A CELL COUNT •/ ^ D DRY WEIGHT 5x10 -10 O 3 -< m O _ 3 3 (Q 3x10 s- 1 1 r 1 1 r 350- ... ^.^wv':-?' ?iUia 5 150- Ul o cr a UJ 0) 3 :j O CO 100- 50- pg/CELL T 1 "I r 12 3 4 5 6 DAYS AFTER SUBCULTURE Fig. 3-5. Arogenate dehydratase activities followed throughout a growth curve of N. silvestris. Activity was followed in samples harvested daily throughout the growth curve shown in Fig. 2-4. A) Activities expressed as units/g dry wt . , B) as units/cell, C) as specific activity. The stippled bars indicate the corresponding activities found in EE cells (average of 10 determinations) , the width of the bars indicating the range of variation. D) total cumulative units of activity per culture. See Fig. 2-4 for meaning of L, E and S. 58 >- > O < UJ < < o >- z UJ o LU t- < z LU O o cr < 0 12 3 4 5 6 7 DAYS AFTER SUBCULTURE 59 activity (Fig. 3-5C) and units of activity per cell (Fig. 3- 5B) between days 3 and 5 (mid-to late-exponential growth reflected a rate of enzyme synthesis that was slower than the growth rate since total units of activity (Fig. 3-5D) continued to increase during that time. From days 5 to 7 the decline in specific activity reflected a net loss of enzyme activity. Discussion C. sorokiniana aromatic-pathway enzyme activities appear to be somewhat lower but consistent with those found in higher plants. The S-protein from N. silvestris must be antigenically different from that of C. sorokiniana, since the S-protein specific antibody did not precipitate the protein from Chlorella. Specific antibodies made to PAT and ADH are now available (see Chapter V) to test cross reactions with these proteins from Chlorella. Although the coincident DQT and SDH activity peak from the DEAE column which eluted protein from C. sorokiniana extract was only purified through two steps, it is conceivable that further purification would yield a bifunctional S-protein like that found in higher plants. Activities from column fractions indicated that DQT activity was about half that seen for SDH, whereas crude extracts indicated that activities were about equal. Possible explanations would be that (i) more than one protein is 60 present in Chlorella for DQT and/or SDH, (ii) some component in crude extract inhibits the SDH activity or (iii) DHS, the product of the reaction is being utilized by another protein. The second possibility may also apply to ADT and ADH since activity was not found in crude extracts but was located in column fractions. Most interesting, is the similarity of PAT in Chlorella to that of higher plants, each having the high optimal temperature for activity. At 65°C, the aminotransferase (s) using the PPY/GLU couple was inactivated. The high specificity of Chlorella for the substrate couple of PPA and GLU was also consistent with higher plant PAT (5, 6) . From these data, it is suggested that C. sorokiniana has an aromatic biosynthetic pathway similar to that established for higher plant chloroplasts. Supportive evidence would include the isolation of Chlorella chloroplasts to determine the location of the aromatic pathway enzymes. The isolation and sequencing of cDNA clones coding for S -protein, PAT and ADH (see Chapter VI) may be useful as probes to clone the appropriate genes in Chlorella. Arogenate dehydratase activities paralleled a similar experiment with respect to the chloroplast isoenzyme of DAHP synthase (Ds-Mn) , where growth of cells and enzyme activities where followed (32) . Both enzymes rise in exponential growth and decline in stationary phase of growth. ADT exhibited an opposite pattern of activity to 61 three other aromatic pathway enzymes, SDH, PAT, and ADH. The three enzymes are known to exist in the chloroplast of higher plants, but the extent to which a fraction of each of the total activities measured might be derived from the cytosol is unknown. All three enzymes exhibited elevated levels of activity in stationary phase and minimal levels of activity in exponential -phase growth (11) . Aromatic -pathway enzymes may be sorting out into two different patterns of expression for reasons yet to be elucidated. Alternatively, since enrichment of Ds-Co (the cytosolic isoenzyme of DAHP synthase) , occurred in stationary phase, rising levels of SDH, PAT and ADH in stationary phase may reflect the expression of cytosolic isoenzymes. If so, the rise in activity of cytosolic isoenzymes exceeds the decrease of the plastidial isoenzyme counterparts. These data suggest that while ADT is present in the chloroplast pathway, it may not function in the cytosol. If so, perhaps prephenate dehydratase of the alternative route (activity, not as yet determined in plants, see Fig. 1-1) , functions in the cytosol to synthesize PHE that may be used extensively for secondary metabolism. 62 CHAPTER IV PURIFICATION OF PROTEINS FROM THE AROMATIC AMINO ACID PATHWAY This chapter includes the separation and purification of the common pathway bifunctional DQT/SDH proteins from Nicotiana silvestris suspension cells. Six to eight steps of column chromatography, including a specific NADP* affinity column have been used. Prephenate aminotransferase and arogenate dehydrogenase were also purified by a series of chromatographic steps . Results DQT/SDH purification In crude extracts, activities for SDH and DQT (specific activity of 61 and 13, respectively) were detected by fluorometric or spectrophotometric assays. The enzyme (s) are not stable at 4°C after several days of storage, unless glycerol and DTT are present. Under these conditions activities remained stable to freeze/thaw, at either -20 or -70°C. Two peaks of activity for the bifunctional S-protein were eluted from a Celite 545 column with an ammonium sulfate gradient (Fig. 4-1) . Elution of the major peak began at about 50% of ammonium sulfate saturation and was C 4-) oV> g d in 13 0) M i-H -H O T) T3 U rC c hi, "S in ^ ^ cnov in d o -H m •-"PS rH *-• O 0) U XI U Q) (0 o . "5 -O CO m fS 0) 0) JJ -H > -U -H (C 4-) U T3 tC Q) o2 H o B 0) CQ n3 4-) >i Si Q) 0) 4-) CO •H o >i Xi m 4: D4 to en o 4-) e o D 0 to I CO QJ Q CO 0) U d o u ^ d to -H o Q CQ w (0 H -H o J-" -^J m W O 0) H (0 OS g -H 4-) O O "5 V^ ^ 14-1 u o 0) 4J m rH 13 e -H d o e e (0 4-1 o d o -H 4-) O 14-1 CQ T) O Xi 4J QJ T) d n5 (0 rH (0 •H ^H 0) 4J to -> "-V-a^ 1 - 0 / 1 1 «,^=P^ T. — 10 20 30 40 50 60 FRACTION NUMBER 70 80 71 Table 4-1. Purification of SI protein from N. silvestris suspension cultured cells, SDH DQT Vol Total Protein Purity Yield Purity- Yield Ratio step ml mg SA fold % SA fold % S/D Crude 540 605 56 1 100 12 1 100 4.7 Celite 643 206 126 2 70 25 2 65 5.0 CM- 52 720 49 412 7 55 94 8 59 4.4 DEAE-1 279 35 561 10 53 113 9 50 5.0 HA 618 8.7 1,515 27 36 257 22 28 5.9 DEAE-2 106 1.95 4,609 82 24 941 78 23 4.9 Aff-1 88 0.42 14,500 259 17 2,342 195 13 6.2 DEAE-3 83 0.16 33,878 605 15 7,365 614 15 4.6 Aff-2 54 0.07 65,877 1176 9 12,923 1077 12 5.1 S/D is shikimate dehydrogenase/dehydroquinase Table 4-2. Purification of S2 protein from N. silvestris suspension cultured cells. SDH DQT Vol Total Protein Purity Yield Purity Yield Ratio step ml mg SA fold % SA fold % S/D Crude 540 605 5 1 100 1 1 100 4.6 Celite 348 97 26 5 68 5 5 64 5.0 CM-52 345 69 31 6 59 8 8 73 4.4 DEAE-1 77 31 66 13 55 14 14 53 5.1 HA 104 5 295 59 43 75 75 53 5.4 DEAE-2 31 1 757 151 34 187 187 34 4.1 Aff-1 15 0.006 4,590 918 7 880 880 7 5.2 S/D is shikimate dehydrogenase/dehydroquinate 72 100 \- 1-100 (0 T 60 E I!t4 CEUTE 545 2.0 1.0 s CM 280 300 320 340 FRACTION NUMBER 360 Fig. 4-4. Celite 545 column chromatography overlap of arogenace dehydrogenase and SP-II. Both SP-II (•) and ADH (O) elute in the 45 to 0% ammonium sulfate gradient (solid line) with overlapping activity peaks. Protein was monitored at A- 290 nm (...) 73 140 120 100 JE 80 E fc> 60 40 20 SDH "~ / -v^^ Hydroxylapatite - r • N. — \ ADH /'"' - - \ 1 1 / \ " — 1™ -^ f— 1 1 :^ , ^V" 600 400 300 g 200 ^ 100 0 20 . 40 60 80 FRACTION NUMBER 100 Fig. 4-5. Hydroxylapatite column chromatography separation of arogenate dehydrogenase and SP-II. The SDH activity (■) was found in the wash fractions and ADH (•) eluted at about 120 mW KPO« . 74 in similar fractions from the NADP* affinity column. Therefore, when ADH was purified by the earlier mentioned procedure an HA column step was included to completely remove SDH activity. Purification of PAT followed the previously demonstrated procedure with a purification of about 250-fold. Discussion The SP-I and SP-II proteins, well separated from each other on the Celite 545 column, were each purified further to about 1100- and 900-fold, respectively, by a series of chromatographic steps. This complete separation was always repeatable under similar conditions of the Celite 545 column step (at least 12 times) . SDH and DQT activities coeluted at an activity ratio of about 5 throughout the entire purification regimen for both bifunctional proteins. These proteins may be isoenzymes from different compartments (chloroplast and cytosol) or they may be isoenzymes residing in different locations within the chloroplast. This second possibility was reported for two DAHP synthase isoenzymes (DS-1 and DS-2) presumably located in chloroplasts of Arabidopsis (2) . Further studies which may elucidate differential properties of the two proteins and localization studies should provide information with respect to possible isoenzyme species and their location within the plant cell. 75 The purified SP-I and SP-II proteins were studied further to characterize both the DQT and the SDH activities in (Chapter V) . The purified SP-I was used to produce specific antibody against the protein (see Chapter VI) and for N- terminal sequencing (see Chapter VII) . Purification of PAT and ADH was completed and then used to produce specific antibodies to each protein for use as probes in cDNA cloning. 76 CHAPTER V PROPERTIES OF THE S- PROTEINS The main focus in my studies of the properties of the two bifunctional proteins was to detect possible differential properties which might suggest that these proteins are isoenzymes. Molecular mass, pH and temperature optima, thermal inactivation, and Km values for both functional domains of the bifunctional proteins, SP-I and SP-II, have been determined. Effects on activity of each protein by various compounds, including PCMB, were also studied. RESULTS Activity peaks on a Celite column A Celite 545 column resolved about 6 peaks of SDH activity when a more shallow gradient of ammonium sulfate was applied than that used during the separation of the S- proteins. Chapter IV (Fig. 5-1) . Dehydroquinase activity was coincident with SDH activities. Fractions of the six peaks containing the enzyme activities were pooled separately, avoiding overlapping fractions as much as possible, and were labeled as shown in Fig. 5-1. The six xi in 4-1 T3 4-) ^ 0 d -H in 0 0 ^ XI u , 4-) rH 4J -H (U MH •H rH 0 > 0) >, -rl u 4J -H > in 4-1 u (0 0 u •H 4-1 U 0 4J Q IW (tJ oV> CO Q) P in in 4-1 0 -H 4-) CO e 0 M MH -H > 0 > -H ^ 4-1 CQ (0 m 4-1 (1) (6 4-1 Q ID •H (1) MH D^rH CO (U (0 ^ Xi :3 CO 14-1 r-{ tJl4-l 0 rQ X B •H 0) •rl 3 (U CQ 4J m -H rH CQ m a ■H 0 MH < 0 IW Qt rH B 0 3 B M 0) CO nS a > •H 0 e -H > o -H c 0 4-1 • 0 0 4J 13 4-1 e 4-1 rH C B o¥> W Q) ■rl (C M in '4' , TJ CTJ H fO (0 0) B 1 ^H rH 0 ID tnx; OJ U 4J XI MH . s •H OS &1 0 S rH 4-1 •H rH •—^ a fa rH c (U 05 grH -H Xi 3 e T) CQ r-{ o to 0 o u rt u vo en 78 O GO CM o CO C\J O SI o o CNJ - § o CO o o CM O o o 00 o CO o o C\J ^ o CO o o C\J T- S lO ^ 31VdinS IMniNOI^l^V lN30d3d DC UJ CD o o < DC LL 79 pooled samples containing DQT/SDH activities were concentrated and examined for possible differential properties, substrate requirements for saturation with SHK and NADP% alternative substrate specificities (i.e., NAD* and QA) , temperature optimum, thermal inactivation, SDS PAGE and activity-stained native PAGE. No significant differences were found between the overlapping peak samples of I to V (data is not shown) . All six protein samples were specific for NADP* and SHK when assayed for SDH activity. Pool sample IV (or perhaps I through V) corresponds to SP-I, and Peak VI corresponded to SP-II. SP-I and SP-II exhibited similar properties except for Mj., Km and activity stained gels. Molecular Mass A difference was seen in the molecular mass of SP-I and SP-II as shown in Fig. 5-2. SP-I migrated slightly ahead of SP-II on SDS gels (A) and on a silver stained SDS gels (B) . Both gels showed bands (or set of bands) at about 59 kD and 40 kD for purified SP-I, and bands at about 61 kD for purified SP-II. A band at 42 kD can be detected when higher concentration of SP-II was used in these experiments. The silver stained gel offered higher resolution and indicated that the affinity purified SP-I also had a set of bands at about 29 kD. Native M,. was determined on a Sephadex G-200 column, ( Fig. 5-3, panels A and B) , to be about 59 kD and 62 kD for SP-I and SP-II, respectively. In crude extract Fig. 5-2. Molecular weight determinations by SDS PAGE and by silver stain of the two S-proteins. A) The SDS gel shows 2 bands for SP-I (584 ng) at molecular weights of about 60,000 and 40,000 in lane 3. Lane 5 has a single detectable band at a molecular weight of about 62,000 for the SP-II (155 ng) . Lanes 1 and 7 are molecular weight markers with MW as indicated. B) The silver staining technique was also used to determine molecular weights of the S-proteins. Lanes 1, 9, and 16 show molecular weight markers. Lanes 2 through 7 represent SP-I and lanes 11 through 14 represent SP-II at various stages of purification as follows: Lane Protein purification step Molecular weight (s) 2 SI 25ul Affinity pure 60, ,000 40, ,000 3 SI lOul Affinity pure 60, ,000 40, ,000 4 SI 25ul DEAE- cellulose 60, ,000 40, ,000 29, ,000 5 SI lOul DEAE- cellulose 60, ,000 40, ,000 29, ,000 6 SI lOul Affinity pure (AB) 60, ,000 40, ,000 29, ,000 7 SI 25ul Affinity pure (AB) 60, ,000 40, ,000 29, ,000 11 S2 25ul Affinity pure (1) 62, ,000 12 S2 lOul Affinity pure (1) 62, 000 13 S2 25ul Affinity pure (2) 62, ,000 14 S2 lOul Affinity pure (2) 62, 000 (AB) is purified enzyme from several runs that were combined, passed through the affinity column a third time and concentrated. For SP-II the (1) represents the purified protein after the first pass through the affinity column and (2) represents combined column runs of SP-II passed a second time through the affinity column and concentrated. 81 94,000 67,000 43,000 B - 30,000 20,100 — 14.400 12 3 4 5 6 7 8 910111213141516 94,000 67,000 43,000 Fig. 5-3. Gel filtration chromatography of S- proteins. A) Concentrated crude extract (■) , pool IV (SP-I) (•) and pool VI (SP-II) (a) (from the shallow gradient Celite 545 column) were passed through a Sephadex G-200 column. The dotted profile represents the three marker enzymes (alcohol dehydrogenase, BSA and carbonic anhydrase) . B) A molecular weight curve based on the column profile from A suggests the molecular weights for the SP-I to be about 59,000 and about 62,000 for SP-II. Crude extract S-protein is suggested to be about 63,000. 83 0.15 - CM 0.05 - A ADH BSA CAD » t » Sephadex G-200 ."., - - - / /a\ * .•-•-'■■■■' J ^y\ V \ ^ \ 1 1 1 ^^'i^ :,/ , , > »sK 1 1 K. - 30 c 201 3 U. - 10 200220 240 260 280 300320340 360 360400420 FRACTIONS O UJ 3 B 2 - ADH o 1 9 - \ BSA SP-II S 7 • V" y' 6 5 - S-Proteins \ — SP-I 4 ^ CAD « 3 o — ^ 2 1 1 1 1 1 100 200 300 VOLUME (mO 400 84 (containing all fractions of the S-protein) , the bifunctional protein was determined to have only one native molecular weight of about 63 kD. Activity stained gels In order to optimize conditions for a native PAGE activity stain gel, various combinations of substrate and dyes were studied with concentrated crude extract, data not shown. All activity stained gels were assayed at optimal conditions as described in Methods (Chapter II) . Activity stained bands required both substrate, SHK and NADP* for visibility. Crude extract revealed 3 bands on native gels after application of the activity stain (Fig. 5-4) . In Fig. 5-5, purified SP-I and SP-II had 2 activity stained bands each. SP-II migrated at a faster rate than did SP-I. pH and temperature optima of S-proteins Both functional domains of the two bifunctional proteins were studied with respect to pH optima (Fig. 5-6) . SDH activity was followed in a pH range of 6.5 to 10 in 200 mM Bis Trispropane buffer and showed optimal activity at pH 9.0 for both proteins. DQT, assayed by the coupled assay method had very low activities when the same buffer was used over the same pH range for both SP-I and SP-II. A pH range from 6.1 to 9.5 in potassium phosphate, Epps or glycine buffers, each at 100 mAf resulted in higher activities and showed optimal activity at pH 7.25 for both proteins. When the three buffers above were used for the SDH assay there Fig. 5-4. Optimal PAGE activity staining for SDH in crude extract. An activity stain of a native polyacrylimide gel was done at optimal conditions when concentrated crude extract of 360 ug was applied to the gel . Three bands are shown after incubation in substrates at concentrations for optimal activity. 86 K I Fig. 5-5. PAGE activity staining of purified SP-I and SP-II. SP-I was added to lanes 2 and 3, and SP-II was added to lanes 1 and 4. Activity staining was done under optimal conditions. A) After incubation for about 15 min, SP-I showed two bands of activity in both lanes. SP-II, at lower concentrations, showed two bands in lane 4, each migrated faster than the SP-I bands. B) The gel was incubated further for about 15 min. The SP-II in lane 1 was then visible. C) After incubation for 1 h, 2 bands were more pronounced for all four lanes. 88 B 1234 1234 1234 Fig. 5-6. S-proteins pH optima for activity. SP-I and SP-II pH optima were determined with purified enzymes (using 4ng and 8ng protein, respectively) in a range from pH 6 to pH 10. Shikimate dehydrogenase activity was determined in the presence of 200 mM Bis Trispropane buffer at the indicated pH values. Dehydroquinate dehydratase activity was determined with 100 mAf potassium phosphate, Epps, or glycine buffer at appropriate pH values for each. 90 91 was no difference in activity levels from that seen with Bis Trispropane buffer. A temperature curve was followed for each activity of the two bifunctional proteins (Fig. 5-7) using optimal assay conditions for activity. Optimal activity occurred at about 40°C for the SDH functional domain and at about 32''C for the DQT functional domain of either protein. Extract of each bifunctional protein was heated to 30°C, 40°C or 50°C for 5 to 25 min, then assayed at room temperature (24°C) as shown in Fig. 5-8. Activity decreased rapidly at all three temperatures, by 10 min of incubation at 50°C no activity was detected for either protein. Even at 3 0°C the enzymes were subject to inactivation. Substrate was shown to protect the functional SDH domain of both enzymes from thermal inactivation (Fig. 5-9) . With the addition of NADP* prior to incubation at 50°C for 5 min, SP-I retained 67% of its SDH activity and SP-II retained about 61% of the SDH activity compared to 8% and 15% SDH activity retained, respectively, when NADP* was not added. SHK was considerably less protective against thermal inactivation with only 18% and 21% retention of SDH activity for SP-I and SP-II, respectively. Without substrate present, SP-I was thermally inactivated to 50% and SP-II to 60% of activity after 2 min at 50°C. Fig. 5-7. Temperature optima for S-proteins. Optimal activity was determined for both SP-I and SP-II purified proteins over a range of temperatures from 24°C to 66°C. A) Dehydroquinate dehydratase activity was determined for the temperature range given. B) Shikimate dehydrogenase activity was determined over the given temperature range . 93 0.25 -A ^_-. v^^ 0.20 - / \^^ DQT1 0.15 - i ^\^ 0.10 - /^ ^\^ DQT2 0.05 1 1 1 1 1 1 1 1 1 1 25 30 35 40 45 50 55 60 65 70 TEMPERATURE (°C) Fig. 5-8. Thermal inactivation of S-proteins. Purified extracts containing the S-proteins were heated at 30°C (•),40°C (a), or 50°C (■) from 5 to 25 min, then assayed at 25°C at saturating substrates. A) SP-I (4 ng protein) was assayed for SDH activities at various temperatures. B) SP-I I (6 ng protein) was assayed for SDH activity at various temperatures. 95 10 15 TIME (min) 20 25 Fig. 5-9. Substrate protection against thermal inactivation of S-proteins. Purified enzyme was incubated at 50°C for 2 or 5 min without substrate added or 5 min with NADP^ or SHK added, assayed at 25°C and compared to unheated extract activity. Conditions for assay were the same as described in Fig. 5-8. SP-I results are shown in panel A and SP-I I results are shown in panel B. 0.30 - 0.25 - 97 B E 0.20 - > 0.15 - 0.10 - 0.05- I L SHK NADP 0 2 5 5 5 SHK NADP 0 2 5 5 5 INACTIVATION AT 50 C (min) 98 Kinetic constants Affinity purified SP-I and SP-II were used to characterize the properties of the two proteins. Both proteins were specific for their substrates and neither enzyme would utilize NAD* as cofactor or quinate as substrate. Saturation of the SDH activity domain of the bifunctional enzyme with substrates, NADP* (at 0.14 mAf) and SHK (at 5.0 mAf), is shown in Fig. 5-10 (A and C) for SP-I and in Fig. 5-11 (A and C) for SP-II (saturating at 0.2 mM NADP* and 4 . 0 mAT SHK. From the double-reciprocal plots in Figs. 5-10 and 5-11 (panels B and D) , the Km values for the SDH domain of both SP-1 and SP-II were found to be 0.02 mM for NADP*, However, different values for shikimate of 0.8 mAf for SP-I and 0.3 6 mAf for SP-II were obtained. Saturation curves and double-reciprocal plots were determined for both proteins (data not shown) when assayed in the forward direction with DHS and NADPH. SP-I had a Km of 0.3 6 mAf with DHS and SP-II had a difference of 0.26 mAf for DHS. The Km value for NADPH was the same for SP-I and SP-II at 0.01 mAf. SP-I and SP-II had different Km values for DQT activity with substrate DHQ (0.07 and 0.04 mM DHQ, respectively, data not shown) . Table 5-1 relates the Km and Vmax values and the ratio of Vmax to Km for each functional domain activity of the two proteins. Fig. 5-10. Saturation curves and double reciprocal plots for SP-I. A) Saturation of shikimate dehydrogenase when substrate, NADP*, was held constant at 0.5 mM and substrate, shikimate, was varied over a range of 0.25 mM to 5 mM. B) A double reciprocal plot of the data obtain- ed in A when SHK was the variable. C) Saturation of SDH when SHK was held constant at 5 mM and NADP* was varied over a range of 0.01 mM to 0 . 5 mM. D) A double recipro- cal plot of the data obtained from B when NADP* was the variable . 100 en 0.1 12 3 4 mM SHIKIMATE ni; c VELOCITY o o < { i S1-Prot9in 1 0.2 = 0.1 1 1 1 1 1 0 0.1 0.2 0.3 0.4 0.5 mM NADP* 7 - B / 6 - / 5 - •/ 4 _ 1 ./ V / 3 2 / / / 1 1 1 1 -1 1 2 1_ [SHK] s ^ 4 ^/^^ 1 V^ <^M w^ V 3 - ^^ 2 'jr^ X ^ 1 1 1 1 1 -40-20 0 20 40 60 80 100 _1 [NADP * J < d) (U nS (U T! 4-) 0) ID a< 0 Cn- -H . Q) o < U 102 AUOOIHA NOI10V3U 8 S S o ^ ♦ 0. 8 - 3 z o CM ? - « UJ Z X CO E AJU0OT3A NOliOVHU 103 Table 5-1. Kinetic parameters for SP-I and SP-II Enzyme Substrate Km (mAf) Vmax (nmol/min) (Vmax/Km) SP-I SDH SHK 0.80 0.60 0.75 NADP 0.02 0.59 29.50 DHS 0.36 0.25 0.69 NADPH 0.01 0.26 26.00 QDT DQH 0.07 1.96 28.00 SP-II SDH SHK 0.36 0.60 1.66 NADP 0.02 0.59 29.50 DHS 0.26 0.27 1.04 NADPH 0.01 0.26 26.00 QDT DQH 0.04 1.47 36.00 Inhibitor effects on enzyme activities Protocatechuic acid (0.5 mM) inhibited SDH activity of both SP-I and SP-II at about 15% at saturating substrate concentrations (5.0 mM SHK and 1.0 mAT NADP^) and about 57% for each at 0.8 mM SHK (SP-I), 0.36 mW SHK (SP-II) and 0.2 mM NADP*. PCA did not inhibit the DQT activities of the two proteins. PCMB was inhibitory to SDH activities for both functional domains and activated the DQT functional domains from both bifunctional proteins as shown in Table 5-2. Neither SP-I or SP-II were effected by additions of 0.5 mAf PHE, TYR, TRP, quinate, and cinnamic acid when added to saturated substrate reaction mixes and assayed for DQT or SDH activities. Metals at 0.2 mM concentrations (calcium, magnesium, manganese, cobalt, zinc, and iron) had no effect on activities of either protein. Incubation of DTT had no effect on any of the activities. EDTA at 10 and 125 vnM had 104 no effect on activity when tested at pH 7.0, 8.6, or 9.5. Table 5-2. Effect of PCMB on Enzyme Activities mM Percent 0 , . 5 mW DTT Percent Protein Enzyme PCMB Inhibition Prevention Reversal Activation SP-1 SDH 0.000 0.0 NA NA NA SDH 0.125 100 100 0.0 NA SDH 0.250 100 100 0.0 NA SDH 0.500 100 95 0.0 NA SDH 1.000 100 81 0.0 NA DQT 0.050 0.0 NA 0.0 50 DQT 0.100 0.0 NA NA 50 SP-II SDH 0.000 0.0 NA NA NA SDH 0.500 100 96 0.0 NA DQT 0.050 0.0 NA NA 55 DISCUSSION A shallow ammonium sulfate gradient on a Celite 545 column revealed about 6 peaks of SDH activities. All SDH activity peaks had DQT activity as well. No differential properties were found between the five overlapping peaks containing SDH and DQT activities which eluted in the gradient from 55% to 45% of (NH4)2S04 saturation. These could be isoforms of SP-I or artifacts of the column chromatography. The sixth peak (also referred to as SP-II) had several differential properties when compared to SP-I which contained all of or some of the five activity peaks from the Celite 545 shallow gradient column profile. A difference of molecular mass of about 3,000 was determined 105 between SP-I (M^ at 59,000 to 60,000) and SP-II (M^ at about 62,000) from gel filtration, SDS and silver stained PAGE. Bands were also present at about 40,000 (SP-I) and at about 42,000 (SP-II). It was not clear what the lower molecular mass bands represent. These bands could be contaminating protein, isoforms of each protein, or proteolytically damaged protein. Activity stained gels showed three bands (each of which may contain two bands for a total of six bands) of activity for SDH from crude extracts, while, purified SP-I had two bands that migrated slower than the two bands of SP-II. The difference in M,., may suggest these proteins are isoenzymes. One possibility, is that SP-I, the smaller M^ protein (the major fraction of activity that was located on the Celite column) may be the mature chloroplast isoenzyme with transit peptide removed and that SP-I, the larger M^ protein (the minor fraction of activity from the column) might be the cytosolic preprotein and isoenzyme that functions in aromatic biosynthesis. A second differential property, that of Km values was observed for SP-I and SP-II. SP-II has greater affinity for SHK, DHS and DHQ, substrates for both functional domain of the bifunctional protein. Both proteins have about the same affinity for cofactor, NADP* or NADPH. These Km values are in the range of Km values found in the literature (listed in Table 1-1) . The difference in affinity between SP-I and SP-II for substrates is also suggestive of differentially located isoenzyme. 106 The Vraax/Km ratio reflects catalytic efficiency of enzymes that catalyze the same reaction in cells. SP-I and SP-II have the same Vmax, but different Kms, therefore, the Vmax/Km ratio suggests that SP-II with the lower Km , is catalytically more efficient than SP-I. PCMB, a potent inhibitor of SDH activity in plant species (48, 53) inhibited both bifunctional proteins, implying that the presence of sulfhydryl groups are critical for catalytic activity of the proteins. This inhibition can be prevented by the addition of thiol reagents, such as DTT, which did, in fact, protect the enzyme from inhibition by PCMB. Most interesting is that PCMB activated activity of the DQT functional domain. My interpretation is that this may reflect physical overlapping of catalytic sites on the protein. Protocatechuic acid, also a known inhibitor of SDH activity (53) inhibited SDH activity of both proteins, but had no effect on the DQT functional domain. PCA is the product of the quinate catabolic system, and its physiological significance as an inhibitor of SDH is unclear. The pH and temperature optima did not indicate any differences between the two S-protein, but were different for the functional domains of the two proteins. The SDH functional domain of the bifunctional proteins had the higher pH and temperature optima. Thermal inactivation studies of the two bifunctional protein showed that the SDH 107 functional domain of each protein rapidly lost activity over time even at 3 0°C and was protected from inactivation by cof actor much better than by substrate. From the data obtained, it appears that SP-II was stabilized to a slightly greater degree than the SP-I (about 6% greater when NADP"^ was present at incubation and about 4% greater for SHK) . Substrate protection of the DQT functional domain of the two proteins with DHQ was not performed at this time. 108 CHAPTER VI SPECIFIC ANTIBODY TO THE B I FUNCTIONAL S- PROTEIN AND TO TWO POST-PREPHENATE PROTEINS Specific antibody was made to the purified bifunctional SP-I, to purified arogenate dehydratase and to purified prephenate aminotransferase. SP-I specific antibody was characterized with respect to both SP-I and SP-II. Results Antibodies made to each protein (SP-I, ADH and PAT) precipitated crude protein preparations or purified antigens on "Ouchterlony" plates. Each enzyme incubated with the appropriate antibody was precipitated and activity was lost. SP-I specific antibody precipitated both SP-I and SP-II antigen on "Ouchterlony" plates at dilutions of antibody up to 32-fold as seen in Fig. 6-1. The precipitant band was more pronounced for the SP-I plate because more highly concentrated protein was available. A saturation curve of inhibition produced by antibody was similar for both SP-I and SP-II when equal activities were established using 0.0084 /xg of SP-I and 0.031 /xg of SP-II (between 0.230 and 0.267 nmol min", respectively). Increasing amounts of c o o s o o XI •H CQ u -H IH -H U 0) 4J CQ 1 O CO n 0) x! Ti to O CQ tC T) S 0) n M tj a, rH '-' 0 J. I CO . CD H >^ (0 M-l i4_i O U o o -H 4-1 OS 4J ^ o Q44J -H "-H CJ i-^ ^co^ CQ CO ^ 0) (U 0) rH X! . 4-» 4-> 0) -U H fO OS S I rH rH IH VO Dj Dj (U O 0 rH H CO H O -H ^^ (U I I I I I I 1 --> 444 423 <-- 1 I ., , I I I KB S K ic a c no 1 o dR I R IV I I I 227 --> 844 603 1 H 1 1 B E 1 H r H 1 N 1 E i a c i 1 d c n n o n n e o d I R d d I R I I V I I V I I I I I I 320 --> 1S3S T r^ — r S B S N a a p d 1 n h e I III I Plasmid B B a a n n I I I I 1376 E S c p o h R I V Fig. 7-3. Sequencing strategy for cDNA encoding the bifunctional protein. Overlapping fragments were obtained when the SP3 cDNA clone was cut with restriction enzymes in the sizes denoted in the Figure. The cDNA clone was first cut with Hind III only, and Hind III plus EcoRV. From this four of six possible fragments were obtained for sequencing, two are shown in the second step of the sequencing strategy. Next the cDNA clone was cut with Ban II and two of three fragment were obtained. Two combinations of RE were used, Ndel plus Sal I, and SphI plus EcdRV. Sequencing was completed for multiple samples of these fragments in both directions . Fig. 7-4. Agarose gels of SP3 cDNA fragments during stages of subcloning. SP3 fragments were visualized on agarose gels after incubation with restriction enzyme (s) . A) The first three lanes contained cDNA fragments cut with Hindlll (from 500 to about 800 bp) , the second set of three lanes contained cDNA fragments cut with Sphl and Sail (about 300 and 1100 bp) and the last set of three lanes contained fragments cut with Banll (about 500,700 and 900 bp) . B) The first three lanes contained cDNA fragments cut with Ndel and EcoRV (about 300, 500 and 900 bp) . C) All lanes show that plasmids carrying the cDNA fragments were transformed into competent cells. The lowest bands represent RNA, the mid bands are the plasmid DNA bands and the top bands are genomic DNA. D) Lanes contain purified plasmid DNA containing cDNA fragments. Bands are of different sizes depending on the size of the ligated fragment. 128 B Mil(# •fin ... tn 129 amino acid sequence is shown in Fig. 7-5. The sequence has an untranslated 3' end of 277 bases following a TAA stop codon and has three to four probable polyadenylation sites prior to the beginning of a nine base poly A-tail. N- terminal amino acid sequence The first 7 N-terminal amino acid residues of purified SP-I were found tobeGEAMTRN. These amino acids corresponded to the deduced amino acid sequence starting at position number 24 (underlined in Fig. 7-5) . Analysis of cDNA coding for the S -protein GCG sequence analysis. The GCG sequence analysis program (26) was used for analysis of the presumed mature S- protein cDNA sequence starting at amino acid residue number 24. The overall isoelectric point was 5.96 for the bifunctional protein (isoelectric points of 5.04 for the AroD functional domain and 6.99 for the AroE functional domain) . A M^ of 60,388 (556 amino acids) was determined for the proposed mature protein (M^ of 28,515, 260 amino acids was determined for the AroD functional domain and M^ of 34,651, 320 amino acids for the AroE functional domain). There were no remarkable differences in amino acid composition when the AroD and AroE domains were compared. The bias of codon usage was similar to that of other higher plant genes, with A and T being favored in the third position of most alternative codons. Rare codons were TGC (one) , CGC (zero) , CCG (zero) , and ACG (two) . 1 Fig. 7-5. Nucleotide and deduced amino acid sequence of cDNA coding for the bifunctional S-protein. The nucleotide sequence of the cDNA encoding the S-protein, containing 2041 bases, is numbered on the right. The deduced 679 amino acid sequence is numbered on the left. The N- terminal sequenced amino acids are in bold and underlined. The stop codon is designated by the asterisk and four possible polyadenylation sites are underlined with hashed marks . CCATTTITGTGCrCTACAAGTrGCTTGCTAATGGACTTQGTAGTGGATTCAGGGGTGAOG CO IPFLCSTSHLLMBLVVDSGVR AAGATGGAOXXWAtWaUkTGACGAaGAACXSAAACACTAA'nTGTGCACCAATaVTCGCA 120 Jl It N B a 1 X « T « ■ BTLICAPIMA GACACAGTO^ATCAAATtnTtavATCTAATGCAAAAtXXrrAAAATTAGTGGTGCTGATCTr ISO 41DTVDQMLNLMQKAI[ISaADL GTTGGGTrTAATGai^'l-lTATATGCl-l'l'lU-'li;GTrGATGAT 900 281 HZAFRSVGFNGVYMPLLVDD (TTTGCAAATTTCTTIVSJGACTTACTCATCTlTAGATrTTGCIXXKTrCAGCTGTAACAATT 960 301VANFFRTYSS LDFAGSAVTI CCTCACAAGGAAGCCATroTTGACTGCTGTGATGAGTTGAATCCTACCGCrAAAGTAATA 1020 321PHKBAIVDCCDBLNPTAKVI OOGGCTGTCAATTGTCTCGTAAGCCGACTCGATGGGAAt.l'H.l'l'im;ni;CAATACAGAC 1080 341GAVHCVVSRLDGKLFGCNTD TATGTDGGTGCAATCTCCGCCATTGAAGAAGCOTTGCAAGGCTCACAGCCTAGTATGTCT 1140 361YVGAISAIBBALQGSQPSMS GGGTXrrCCCTTACCTGOTAAATTATTTGTGGTCATnXnXXnXXnXXXrGCTWXauUJGCA 1200 aaiGSPLAGKLFVyiGAGGAGKA CrrGCTTATOGTGCAAAGGAAAAGGGGGCTCGGGTGGTGATroCTAACCGTACCTATGAA 1260 401bAYGAXiKGARVVIANRTYB CGAGCGAGAGAAl. I'lU- mATGTACTTGGAGGTCAGGC'l'l'm IVlX-'l'lUACGAGCTTAGC 1320 421 RARBLADVVOGQALSLDBLS AATTTCCATCCAGAAAATGACATGATTtrrTGCAAATACCACCrCCATTGOCATOCAACCA 1380 441 NFHPIHDHILANTTSlaMQP AAGX FFLC8TSMLL Sce-Arol . . . . Y I A T . . Eni-AroM V A T G Q Ncr-Qa-lS L P K G T Eni-QutR L A T G N KTVTVKDLV IGT KTVTVKNLI IGE NVLTIKOVS IGE KPVIVKNVR IGE ELVVDSOVR KHE T D P F V B . . . S A PELRNQLSP a A P K . . . 0 M P K . . . s M P K . . . a N P K . . . a E A H T K ■ .GVRBIEIPS .SLSIIK.EG FPLASVPVEO FPLHMQPISS E T L IVSLMA KDIASVKSEA 34 IVSLIIG RDINSVKAEA 34 IIPLKG KTEKQILMEA 34 VVPIVA PTAEDILAEA 34 CAPIKA DTVDQMLHLH 50 GRSAFV.CLT FDD EHSPFA.SLT LPD RRFTYALALP VSA RKFTYAATVP ISH T E 0 T E N 1092 R E A G D I 1068 L D K G V D 328 L E N D V D 328 Eco-Aron LAYREADF Sty-AroD LAYREATF Bsu-AroD EAVKLLNP Efa-AroD TASQTLDC Hta-AroD«E QEAXISOA dIi D I D I D L D L Sce-Arol Eni-AroH Ncr-Qa-lS Eni-QutR LTPICYOCE LEBVCVOSD IQELDVaVD lEELESTAD w ■g w H W V 5. vrsiv l[rJv I I V L K I HYAD LSNVESVM HFMD lASTQSVL VPEK AMDREAVT VYEN VADFSDVC SLIS FHPQSDID HLAN YSADPVS LLKD PAS N NNIPSVDYVV DLAT SESGPTSPLG LAPHRASBIS VSA. ...APSARLG TBSHLADSIS A A A X t L R B T M 72 T A A R V I R D A M 72 It L I S K L R K S L 72 N L S Q Q V M E R L 72 T I I K Q • 83 K 0 L S I L fRl K A T 1129 B Q L S F L R s R 1111 R V V G E I R R D T 378 H T V A T V R R N I 374 Eco-AroD Sty-AroD Bsu-AroD Efa-AroD P B K P L P D I P L E D K L P G Q K Hta-AroD'E S P L P T Sce-Arol D S I Eni-AroM V T L Ncr-Qa-lS V . I Eni-QutR I . V L F TlF . . fH L F T F . . E L F T F . . R L L T P . . H lJf[tJt . . R fpl I |T| p I I p I I p M lli EQ AIST.EAYIA EQ TITT.QHYLT SH EMDE.SSYLA EM APSE.ENYPA QY AODE.VSRLD F T V . F T I . . R T M K Q . H T Q S Q S S Q G N P R P PDEEFXTLRE PDNAHDAALE L H V . Y H V E . . V F P E S S V F P D R A S A h Y P L EKALLALYMT RRS . DASYLE LHRAAIDSOL LNRAAIDSOL LLBSAIQTKD LYHELVKKOA ALKVAMELOA LYDIALKNQ. LYELAFRSO . YLNHALRLA . LVLHGLRLO . V 0 M I D L E L V D M I D L E L I D L I D I E L L D L L D I E L D y I D V E L F T 119 P T 119 F S 119 P A 119 . X 128 V B F LIDlL E L T L 1176 C E F v[dJl D I a F 1158 PDYLTVDLGL 423 PEFVTVDLSF 421 Eco-AroD GDDQVKETVA Sty-AroD GDADVKATVD Bsu-AroD GDANVKALVS Efa-AroD NPLAADTLIH Mta-AroO'E AIDEFETALH Sce-Arol PTDIQYEVI . Eni-AroM PEDMLRAVT . Ncr-Qa-lS DSGLLGQLT. Eni-QutR EDSILSQII . YAHAHDVIVV YAHAHNVYVV LAEENNVYVV EAKKAGIKIV QHXSAXCXVI N X R [g] N T fxl I IT] E M K G F S K 1 I T V Q G T T X V I G T K [gJ S S X V LlI M S N fSl M s N H M s N E L c N H V s S [si D F H K D F H Q D F E K D F Q K H Y D H T P T P T P T P T P G S H H D F Q . G L A s h[hJd P K . G E GNKQLABVNS GHYSQTQPPP E A E ■j S A E VXD S Q E S S E E. Y S [w] L S W P R w Q G w I I A M V S IIS I V A L O H R L R K M ^ S F D fXl R L R K M Q A L Q A R L R K M Q D L a A R L R Q M Q M R Q A L V A R I ^ A s a aJ D D A E H B H R P N 0 A A N M S W I K P Y N K A G D P S W L 0 A Y E K A S D P E Y E A I Y E R A 166 166 166 166 175 L T L D V 1223 L B Y O . 1204 Q N T O C 471 K K L O C 469 ECo-AroD Sty-AroD Bsu-AroD Efa-AroD Ht«-AroO*E Sce-Arol Eni-AroM Ncr-Qa-lS Eni-QutR D f P Y I pCl D P I A H P M A D C I A D I. V s r A L M P Q P Q V M P N V M P Q T T A L V VfXlF V G T I l[£jL V G V L V R L T R P M V R L T Q P S T S S K H D T G DAT DIM N P N I N P T I D V D V D V D V D V D iT O N D N D N L T L L T L L T I, L T L A R V L E L T A L T D I F A V L A L T A T A T A T A T I T LEMQEQYA LEMQQHYA YTMKTIYA NEMYTHYA V as R D T H K N . . W A A E A HVAVBAVGG RHQIKTLPG D D R S V Q V p I P V p I p I p I P L P h P F P V I I T II S M V I T H S II I I T H S M I V T M S H I I A[lljv[lf A K T A K A A T 0 Q L a E G V I G V I G L I S M I G L II G S I G 0 L 0 R T G R QL S R L A 215 S R L A 215 S R L S 215 S R V T 215 S R 1 — I L 219 S RlV L 1268 S R I L 1252 S M C P 521 S C C F 519 Eco-AroD Sty-AroD Bsu-AroD Efa-AroD Nta-AroD«I Sce-Arol Eni -AroM Ncr-Qa-lS Eni-QutR G E V F G S A A T F G E V F a S A A T F G E V T a SAC T F G Q L T a SAL I F CPE T 0 G Y I, T F N N V L T P V N G F M T P V N E I L T P V N P V L T P V G A G A P A G S V K X A V K Q A G E E A Q Q A OTLEVSXV S A S A S A S A S A p G e p G g P 0 Q P G Q P 0 Q TSDLLPNS AfAl SHPSLPPK aIaJ TP. .VPTKED IPRSLISQSG s V N D [lI A V N D L P V S E L S V Q V L T I X D jj P G Q L IP O Q L A I GIL T K gIl T V A Q I S A T E I R N P A H TVLTILHQA 252 SVLMILHNA 252 SVLDILHKNT RG 256 ■YLKTFEQNK 253 MIYEFRQLGP H 206 KMYTSMGGIB 1305 KGLSLMGEIK 1289 YLQPPLTALB ATQ 559 ...PSITIQE AQBALYSSPV 557 Fig. 7-7. Dendrogram of dehydroquinase homologues . The dendrogram is based upon the multiple alignment which includes the nine designated proteins explained in the legend of Fig. 7-6. 137 E. coli AroD S. typhimurium AroD B. subtilus AroD E. faecalis AroD N. tabacwn AroD'E S. cerivisiae Arol E. nidulans AroM N. crassa Qa-lS E. nidulans QutR -^ 138 Fig, 7-7. The latter shows two clusters, with Nta-AroD«E grouping with the mono functional proteins of the prokaryotes: E. coli, Salmonella typhimurium, Bacillus subtilis, and Enterococcus faecalis. Thus, the bifunctional plant enzyme is evolutionarily closer to the prokaryote monofunctional proteins than to the AroD domains of the pentafunctional proteins present in the eukaryotes, yeast (Arol) and fungi (AroM) . The proteins in the multiple alignment shown in Fig. 7- 6 are separated between the two clusters that are shown in Fig. 7-7 for clarity. This alignment shows the conserved residues within each cluster, as well as those conserved throughout both clusters. Three amino acids shown to be important catalytic residues for the E. coli AroD protein, HIS (25) , MET (47) , and LYS (20) are marked with asterisks, and these are conserved in all proteins having AroD catalytic activity. It has been suggested that the repressor proteins from Neurospora crassa (Qa-lS) and from Emericella nidulans (QutR) evolved from three of the five domains of the pentafunctional protein corresponding to dehydroquinase , shikimate dehydrogenase, and shikimate kinase (38) . These retained the ability to bind what previously were substrate molecules to function in a new role as regulatory agents. Thus, critical residues such as those marked by asterisks were altered to retain binding but to lose catalysis. Note that nine other residue positions 139 are completely conserved in the catalytic proteins, but not in the two regulatory proteins. This implies that some or all of these residues may be important for catalysis. Six residues are absolutely conserved for all nine proteins. Homolocrv relationships of the SDH domain A multiple alignment is shown in Fig. 7-8 of the AroE domain of the S-protein with its homologues. The corresponding dendrogram is shown in Fig. 7-9. Again two evolutionary clusters are seen in the dendrogram, and the AroE domain clusters with the monofunctional AroE protein of E. coli, rather than with the AroE domains of the pentafunctional proteins (Arol and AroM) or with the repressor proteins (Qa-lS and QutR) . The Nicotiana AroD domain is also homologous with the catabolic quinate dehydrogenase of yeast and fungi, which are monofunctional and specific for NAD*. However, the latter (QutB and Qa-3) are evolutionarily closer to the repressor proteins (Qa-lS and QutR) than to the shikimate dehydrogenase domain of the plant bifunctional S-protein. It is surprising that the catabolic QDH exhibits homology with biosynthetic SDH while the catabolic dehydroquinases display no obvious homology with biosynthetic dehydroquinases (32, 35) . This is all the more striking in that the former differ in substrate specificity, whereas the latter do not. One might also have expected the catabolic dehydroshikimate dehydratase to be homologous with the biosynthetic dehydroquinases or Fig. 7-8. Multiple amino acid alignment of the shikimate dehydrogenase domain of AroD»E and its homologues. Nine pro- teins have been aligned and conserved regions are boxed. The probable domain for the NADPH binding site is underlined. The amino acid residue numbers for each sequence are on the right of the figure: Designation Organism Protein Accession Number Eni-QutB EmericeIJa nidulans Ncr-Qa-3 Neurospora crassa Ncr-Qa-lS Neurospora crassa Eni-QutR Ewericella nidulans Sce-Arol Saccharomyces cerivisiae Eni-AroM Emericella nidulans Eco-AroE Escherichia coJi Nta-AroD«E Nicotiana tabacum Eco-URF Escherichia coli quinate dehydrogenase P25415 quinate dehydrogenase P11635 quinate repressor protein P11637 quinate repressor protein M59935 arom pentafunctional protein P08566 arom pentafunctional protein P07547 dehydroquinase P15770 bifunctional S-protein unidentified reading frame P28244 141 Eni-QutB Ncr-Qa-3 Ncr-Qa-lS Eni-QutR Sce-Arol Eni - AroM MEPITIPTDR LTSTPDITPY D D Eco-AroE Ht*-AroO«I D Eco-ORP D G V T R H P M K P H Q P K B P K K M B T Y A V T K I r a I Y 0 F 0 F 0 F 0 V a F O F a I 0 HB R N 8 L 8 P P L H A A S M 3 P I. I. H G Y s L 8 P A M H T Y s L 8 P A M H G E s R 8 P I L H s g s R 8 P A L [ill A H S K 8 P F I H S H S X S P L L T QTVYNALOLH WTQIPLSTAT 50 SIVYSHLSLH W A Q L R L D . . . 73 ■ AXLKACaiP HHYKPtSTAN eo8 ■ AAFKVROMP HIYRIHQSPT 599 ■ TGYEILOLP HKPDKFETES 1345 ■ TLFAQVOLP HNYTRLETTN 1329 QQFAQQLNIB H P Y G R VfLlA P I 40 ■ lAFRSVOFH OVYMPLL. .V 298 Eni-QutB Ncr-Oa-3 Ncr-Qa-lS Eni-QutR Sce-Arol Eni-AroM EcoAroB Nta-AroD>I Eco-URF GTSFTRSPB S P S I G T L R E . . . L R G I ■ Y . . . A Q L V K B K . . A Q D V Q B . . . NOFIHTLNAP DDVAHPrXTT STFLSSVRSN PLFL.QLAQH V I S D L V B N L L D G N F I R 8 :[S P K ■f V fol P D Y 0 P 0 A 0 P N G 0 K N G a P D L G 0 A G G K 6 L D T A [oj 8 S V A S V S V T S V AAV APR m T M T H G L S L T I H H T V T I S M p H Y p H p F p Y p L s L ■p F p H ll N li V A I M P H L DDL T E D A R Q A 100 V A I I P H L DHI.TPECRDV lie V B I I S L T HSLSRHAXAI 648 T E V I P L L HSMSPHARAI 639 L D I M Q Y M DELTDAAXVI 1387 L D I M P L L D K V A A B A E I I 1369 I E A P A R A DEL T E R A A L A 82 I A Z V D C C D I L ■ P T A X V I 338 Q L A C E Y V D B L T P A A X L V 22 Eni-QutB Ncr-Qa-3 Ncr-Qa-lSc Eni-QutR Sce-Arol Eni-AroM Eco-AroB Kta-AroD>I Eco-URF a A C H T 0 A C H T a A V ■ T 0 A V ■ T a A V > T 0 A V H T a A V H T a A V ■ c a A I « T I Y L S K L P L K T L I P V R L I P I R V I P L G I I P V S L K R I E 8 X L I V N . D D HLTADGGIPD NI,..EGSTDN N BVSMFNNISQ ALDLEKN. .R K T G i: D D G K T 0 Y A T G R R L Y A G A V R A L AGP I K G L K . . . F K N T P S R L G R L 0 K L G Y L V G T 11 T D C I 0 c G A a T D V I a Y G E H T D H I a H G D ■ T D W I a K G D ■ T D W L a V 0 R ■ T D W Q 0 L a D BID G V a F 0 C » T D Y V 0 R 0 Y H T D G T 0 I R E A 131 V R E S 148 I R A c 698 I G I c 685 I R N A 1414 M I L S 1401 L L 8 D 109 A I 8 A 365 H I R A 48 Eni-QutB LLQGSPNGAE H F K 0 . K P A . . Ncr-Qa-3 FVQNVSDPAR V Y E S . R P X . . Ncr-Qa-lS LRR . GLSPAN AVRSTSTG. . Eni-QutR IRR.GLSPAN AIRP8TTG. . Sce-Arol L I N N G V . . . P EYVOHTAG. . Eni -AroM LRKAGVYGPK RXDQBQSA. . Eco-AroB LXR.LSFIRP 0 L R ■ta-AroO'I IIIALQ08QP 8iisaspLAax Eco-URF I K 1 . SOFDIKSX L I V G 0 G G T A L V I G 0 G 0 A A L V I G A G G M A L I I G A G 0 M A L V I G A G 0 T S LkJ V V [gJ a [go T A . I L L I G A 0 0 A I F V V I 0 A 0 0 A T M V L L G A 0 0 A R T A I pY R S A V Y R AAV Y R A G I Y R A A L Y LeJ A A I [yJ V L A L A M A M A L A L R K W H K H L Q . I H . H S . H N . SRGV.LLPLL SXAL.ATGAX STAIGAQGAI L [g] L G L G L G L G M [gJ S K I Y I V H 177 ATDIYLVN 194 VKKILIFK 743 VQNIFIWB 730 CKKIFIia 1456 YSPIYIVG 1447 SLDCAVTI Tir 149 IXOAKVVI A a 414 EGLKEIKL plal 89 Eni-QutB S D A K E V X A I L A Ncr-Qa-3 DKSBVDAVl A Ncr-Qa-lS TFANAIKLV L Eni-QutR TVANAIKLA 0 Sce-Arol TTSKLKPLI I Eni-AroM TPSKLXNMV S Eco-AroE T . . .VSR rsi B X ata-AroD«X T . . . Y I R A X K Eco-URF R D E F F D K A L A E DKQ RNPSPQVALV E CTE RGYGDR..LV HFBNLLVRD ALPLLSTOPR SHDNTCFHII HYMR LNLCTLOGS GSASYTIHVL SL PSEFNIIGI SF PSSYNIRIV L A K . . LAD.. F A Q A L . . . . L F A H T G . . . . V V O 0 . . MKTPIVSSRS S I Q A L . . Q A L P I S P LfSlM D XfLl L 8 L D XL i[sJk p l[lJ PVSDPSAAAT 212 HVASVXQAEG 227 RSRDDPLPEN 793 KSLQESWPAN 774 ES.TKSIEEI 1488 ES.PSSFESV 1478 EG. .HXFDLI 184 saFBPxaoai 449 KPWIP..PTF 133 Eni-QutB Ncr-Qa-3 Ncr-Qa-lS Eni-QutR Sce-Arol Eni-AroM L E L E F K Y K K E P H A G a Q H p p p p V V A V V S G G A I V A C T M I V S C I I V V S G G V A V S C . V A I G T 1 I I I V I P P P P P P NYPPQTEE DFPPKTEK T H T . V D N T A H R . I G D Q ADKPLDDE ADQPIDPT EIRARETLRL BMLVRRIVBT PDPEFTVPLH PAPNFQLPPQ LLSKLERFLV MRETLCHMFE Eco-AroE I . L A L T a a A I G [tIb sFo] . . Nta-AroD*X TSIO Eco-URF TKVO FLNRQTHEKD QG VI 256 FLMKE....E KG AM 267 WLDNPTGG IV 831 WIBSPTGG VV 813 KGAHAA FVPTL 1529 RAQBADABAV KAIEHAPRIL 1525 .ISGDIPAIP SSLIHPGIYC 210 .HQPXVDDTP ..IFXXALXT 474 .HKP. . . .LR MNHWLMISIC 156 Eni-QutB Ncr-Qa-3 Ncr-Qa-lS Eni-QutR Sce-Arol Eni-AroM B M C lYl E M C Y E L D Y D L A Y E A A Y B H A Y Eco-AroE Y D M F Y Q K G K T ata-AroD>I Y S L V F D A V Y I Eco-URF Y I R D F W S L K L T D T B L E P . . . F L A H C E PXITILLXXA A C I T X I . . . . Q D A E H E A H R S H R D K Y D S . ORGS H C S O R 0 G O 0 0 X V I L [o] Q V I L 0 V A H D 0 A A L D 0 H V V P 0 I T I P 0 R N A D s X I V T [o| S X A T I A L D L L D V S Q M L X V GfalL V x[iiJf I Q AlA a a|y Q Q Q Q Q A F Q T L L H HraiV X X F t[s]l D V T L R A R A K G L A L P A 8 303 314 881 862 1576 1567 257 524 172 Eni-QutB VSBPGLVEKV Ncr-Qa-3 Y C O G Q L N . . . Ncr-Qa-lS PRRLMRREVL Eni-QutR PRRLMRTVIL Sce-Arol P F K A . . . .IF QAFVAQTIAB R S K S N L RAYPDDQAKS QEYKEBBQGB D A V T K E . . . . HTAQ. LQPR BYDQSAMRTR LNGIATQIS . LENLDGQPM . 329 321 918 901 1588 Fig. 7-9. Dendrogram of shikimate dehyrogenase homologous. The dendrogram is based on the multiple amino acid alignment and includes the nine proteins defined in the legend of Fig. 7-8. 143 E. nidulajis QutB N. crassa Qa-3 N. crassa Qa-lS E. niduleuis QutR S. cerivisiae Arol E. nidulans AroM E. coli AroE N. Cabacum AroO*E E. coli URF 144 catabolic dehydroquinases . Table 7-2 shows each pairwise comparison of N. tabacum AroD«E with its homologues (Gap program) . Table 7-2. Pairwise comparisons of S-protein with its homologues . Percent Organism DQT SDH and Gene Identi ty Similari ■ty Identi ty Similarity Sce-Arol 29 53 28 56 Eni-AroM 24 52 27 51 Eco-AroD 30 52 - - Eco-AroE - - 31 55 Sty-AroD 29 51 - - Efa-AroD 34 54 - - Bsu-AroD 32 53 - _ Ncr-Qa-lS 22 44 30 53 Eni-QutR 21 46 26 47 Eni-QutB - - 28 53 Ncr-Qa-3 - - 26 50 Eco-URF - - 36 61 StU-MDH - - 23 51 Nta-CDH - - 21 47 NAD (P)H-binding proteins often share a motif (Rossman fold) dominated by glycine or other residues with small sidechains such as alanine (72) . Table 7-3 shows a comparison of this region in the organisms compared in this dissertation with a number of others recovered by the GCG Blast program. The S-protein fits the NADPH binding motif perfectly with GxGxxAxxxA (72) . A survey of some chloroplast transit peptides -•1 145 Table 7-3. NAD{P)H Binding Motif of Enzymes'. Organism cDNA or Gene Product Conserved Region GxGxxGG GxGxxVxxxA Accession GxGxxAxxxA Number E. nidulans N. crassa E. nidulans N. crassa E. nidulans S. cerevisiae E. coli E. coli N. tabacum S. tuberosum N. tabacum N. tabacum H. vulgare T. repens C. roseus T. aestivum N. silvestris H. sapiens H. sapiens M. musculus P. stipitis S. cerevisiae K. marxianus C. synechocystis Quinate repressor (QutR) Quinate repressor (Qa-lS) Quinate dehydrogenase (QutB) Quinate dehydrogenase (Qa-3) Shikimate dehydrogenase (AroM) Shikimate dehydrogenase (Arol) Shikimate dehydrogenase (AroE) unidentified reading frame S-protein {AroD»E) Malate dehydrogenase Cinnamyl -Alcohol dehydrogenase 1 Cinnamyl- Alcohol dehydrogenase2 Alcohol dehydrogenasel Alcohol dehydrogenasel S-Adenosylhomocysteinase S-Adenosylhomocysteinase S-Adenosylhomocysteinase Alcohol dehydrogenase gamma Glutathione reductase Lactate dehydrogenase D-Xylulose reductase Succinate dehydrogenase Alcohol dehydrogenasel Ketolate reductoisomerase STTGLIIGAGGMARAGIYAM M59935 TSTGLVIGAGGMARAAVYAM P11637 GKPALIVGGGGTARTAIYVL P25415 SRPALVIGGGGAARSAVYAL P1163 5 EQSALWGGGGTARAALYAL P07547 HTAGLVIGAGGTSRAALYAL P08566 GLRILLIGAGGASRGVLLPL P15770 GKTMVLLGAGGASTAIGAQG P28244 GKLFWIGAGGAGKALAYGA KMKIWA6AGSAGIGVLNAA Z23023 GFRGGILGLGGVGHMGVKIA P3 0360 GLRGGILGLGGVGHMGVKIA P30359 GSTVAIFGLGAVGLAAAEGA P05336 GSSVAIFGLGAVGLAAAEGA P13603 GKVAWAGYGDVGKGCAAAL P35007 GKVAWCGYGDVGKGCAAAL P32112 GKVALVAGYGDVGKGCAAAL D16138 GSTCAVFGLGGVGLSWMGC P00326 SYDYLVIGGGSGGLASARRA S08 979 NNKITWGVGQVGMACAISI P16125 GDYVAVFGAGPVGLLAAAVA P22144 WIGAGGA6LRAAFGL Q00711 TKLPLVGGHEGAGVWAMGE P 2 0 3 6 9 GKTVAIIGYGSQGHAHALNL A4703 7 E. coli Threonine dehydrogenase E. coli Glutamate synthase T. brucei Phosphogluconate DH T. brockii NADH oxidase P. putida Formaldehyde DH GEDVLVSGAGPIGIMAAAVA P07913 GKKVAIIGAGPAGLACADVL B2 9617 WGLGVMGANLALNI P31072 KKWWGGGPAGMQAAITA P32382 GSTVYVAGAGPVGLAAAASA D21201 "Motifs were taken from Scrutton et al . , 1990. The first nine set of organisms are those used in the SDH multiple allignment of Fig. 7-8. :d 146 established in the literature is shown in Table 7-4. The transit peptides vary from 34 to 88 amino acid residues. The sequence context around the translation start (AUG) is proposed to most often be AACAAUGGC in plant genes (54) . Included in this survey are the seven known sequences for aromatic pathway proteins. From this selection in Table 7- 4, only one of sixteen, chorismate synthase in barley (aromatic pathway protein) has this proposed conserved region. The cleavage site often exhibits the pattern (V/I)- X- (A/C) iA, with R frequently at position -2 or between -6 to -10 (33) . Of the seven aromatic pathway proteins, shikimate kinase in tomato conforms to the motif before the cleavage point and five of the remaining nine sequences conform to the cleavage point (two of these also have the proposed ALA after the cleavage point) . The S-protein does not fit this cleavage site motif. The overall features of transit peptides, show that they are rich in hydroxylated amino acids, SER and THR (20-35%) , small hydrophobic amino acids such as VAL and ALA, not especially rich in basic amino acids (but do have a net positive charge) , and usually have only one or two acidic groups (45) . The aromatic pathway protein transit peptides follow these features, being especially rich in SER. The S-protein truncated transit peptide (23 residues) has 17% SER (3) and THR (1), 35% hydrophobic residues, 0.09% basic residues {R (ARG) and K (LYS) near the cleavage point} and 13% acidic residues. 147 Table 7-4. Transit Peptides of Aromatic Pathway proteins and of other Plant Proteins. Enzyme Plant Start Transit Peptide- Amino Acid Number Mature Protein DAHPsyn agcaatggc potato MALSSTSTTNSLLPNRSLVQNQPLLPSPLKNAFFSNNSTKTVRFVQPISAVHSSDSNKIPIVSDKPSKSSPPAA EPSPsyn agaaatggc tomato MAQISSMAQGIQTLSLNSSNLSKTQKGPLVSNSLFFGSKKLTQISAKSLGVFKKDSVLRWRKSSFRISASVATAE EPSPsyn aagaatggc petunia MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLLNSANSMLVLKKDSIFMQKFCSFRISASVATAQ EPSPsyn tccgatggc cruc . weed MAQVSRICNGVQNPSLISNKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIGSELRPLKVMSSVSTAE CHAsyn aacaatggc barley MASSLSTKPFLSGSRRRSTTDGSGWSYFQTSDLRQLSNQSVQISVRRQTAPLKLWQASG SHKkin gactatgga tomato MEARVSQSLQLSSWINSDKWRKPSGLLRFSEKWNEKPRHRWVSCHLQPRKAAHSDRRVQLKVSC TRPsyn agtaatggc cruc . weed MAASGTSATFRASVSSAPSSSSQLTHLKSPFKAVKYTPLPSSRSKSSSFSVSCTIAKDPPVLMAAGSDPALW ASPat aaacatggc soybean MASSFLSAASHAVSPSCSLSTTHKGKPMLGGNTLRFHKGPWSFSSSRSRGRISMAVAVNV GSAat catcatggc barley MAGAAAAVASGISIRPVAAPKISRAPRSRSWRA L12ribp tacaatggc tobacco MASTLSTITLRSPSPSTASSTHASIPFPKKALEFPIRTPKLHHRRAT IPMdh tgaaatggc rape MAAALQTNIRPVKFPATLRALTKQSSPAPFRVRC PCPoxre agtaatggc pea MALQTASMLPASFSIPKEGKIGASLKDSTLFGVSSLSDSLKGDFTSSALRCKRELRQKVGAVRA FEDred cgccatggc spinach MTTAVTAAVSFPSTKTTSLSARSSSVISPALISYKKVPLYYRNVSATGKMGPIRA FBPald taagatggc spinach MASASLLKTSPVLDNPGFLKGQTLRIPSVAGVRFTPSGSSSLTVRA CARanhy accaatgtc pea MSTSSINGFSLSSLSPAKTSTKRTTLRPFVFASLNTSSSSSSSSTFPSLIQDKPVFASSS G3Pacyt gggatgac pea MTDSFAHCASHINYRHKMKTMFIFSTPCCSPSTAFFSPFRASNSKPLRSTLSLRSSISSSSITSTSHCSLAFNI IVKHKEKNWSANMT 72 TATTAP. KPSEIV. 65 SPQNVQ 72 QRPDSF 60 SRFEGI 34 AVSIDE 53 FLRPLA 33 AAASPG 64 ETAAPA QIASDV. . S-PROT tobacco not known PFLCSTSWLLMELWDSGVRKME 148 SUMMARY In conclusion, with the cloning of the aroD^aroE cDNA encoding the S-protein (the first to be cloned and sequenced in higher plants) , other studies may now be undertaken. First of all, a complete sequence is desirable, since it is proposed that the 5' region has been truncated. This may be accomplished by obtaining mRNA from Nicotiana tabacum plants for primer extension. Other studies may include cloning of the gene from a genomic library, determining the number of gene copies in Silvestris or in other plant species by use of Southern blots, performing site directed mutagenesis or chemical treatment to study the active site residues, use of the cloned cDNA coding for the S-protein as a probe to obtain the cDNA from other plants and to determine what the cytosolic pathway has with respect to dehydroquinase and shikimate dehydrogenase. The probable location of the bifunctional gene product in the chloroplast remains to be proven. Clarification of the relationship of SP-I and SP-II is needed. The possibilities are: (i) that one is an artifact of limited proteolysis, (ii) that one is an active, uncleaved preprotein in the cytosol, (iii) or that they are separately subcompartmented in the chloroplast . 149 REFERENCES 1. Anton, I. A, and J. R. Coggins. 1988. Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase. Biochem J. 249:319-326. 2. Bentley, R. 1990. The shikimate pathway- -a tree with many branches. Crit . Rev. Biochem. Mol . Biol. 25:307-384. 3. Berlyn, M. B., R. L. Last, and G. R. Fink. 1989. A gene encoding the tryptophan synthase jS subunit of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 86:4604-4608. 4. Bonner, C. A., R. S. Fischer, S. Ahmad and R. A. Jensen. 1990. Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact. Appl . Environ. Microbiol. 56:3741-3747. 5. Bonner, C. A., and R. A. Jensen. 1985. Novel features of prephenate aminotransferase from cell cultures of Nicotiana silvestris. Arch. Biochem. Biophys. 238:237-246. 6. Bonner, C. A., and R. A. Jensen. 1987. A selective assay for prephenate aminotransferase activity in suspension- cultured cells of Nicotiana silvestris. Planta. 172:417-423. 7. Bonner, C. A., and R. A. Jensen. 1987. Prephenate aminotransferase. Methods Enzymol . 142:479-487. 8. Bonner, C. A. and R. A. Jensen. 1987. Arogenate dehydrogenase. Methods Enzymol. 142:488-4 94. 9. Bonner, C. A., and R. A. Jensen. 1991. Response of arogenate dehydratase to changing physiological states of growth in suspension cultures of Nicotiana silvestris. Plant Science. 74:229-234. 10. Bonner, C. A., C. Kenyon, and R. A. Jensen. 1988. Physiological and biochemical characterization of a "-^ suspension-culture system for sustained exponential r| growth of Nicotiana silvestris. Physiol. Plant. 74:1-10, i 150 12. Bonner, C. A., A. M. Rodrigues, J. A. Miller and R, A. Jensen. 1992. Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture. Physiol. Plant. 84:319-328. 11. Bonner, C. A., J. Vrba and R. A. Jensen. 1988. Response of aromatic -pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris. Physiol. Plant. 73:451-456. 13. Boudet, A. M. , and R. Lecussan. 1974. Generatite' de 1' association (5-dehydroquinate hydro-lyase, shikimate : NADP* oxidoreductase) chez les veg etaux susperieurs . Planta. 119:71-79. 14. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72:248-254. 15. Brotherton, J. E., R. M. Hauptmann and J. M. Widholm. 1986. Anthranilate synthase forms in plants and cultured cells of Nicotiana tabacum L. Planta. 168:214-221. 16. Byng, G. S., J. F. Kane, and R. A. Jensen. 1982. Diversity in the routing and regulation of complex biochemical pathways as indicators of microbial relatedness. Grit. Rev. Microbiol. 9:227-252. 17. Charles, I. J., J. W. Keyte, W. J. Brammar, M. Smith and A. R. Hawkins. 1986. The isolation and nucleotide sequence of the complex AROAf locus of Aspergillus nidulans. Nucleic Acid Res. 14:2201-2213. 18. Chaudhuri, S., I. A. Anton, and J, R. Coggins . 1987. Shikimate dehydrogenase from Escherichia coli. Methods Enzymol. 142:315-320. 19. Ghaudhuri, S., K. Duncan, and J. R. Goggins . 1987. 3-dehydroquinate dehydratase from Escherichia coli. Methods in Enzymology. 142:320-324. 20. Chaudhuri, S., K. Duncan, L. D. Graham and J. R. Coggins. 1991. Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases . Biochem J. 275:1-6. 151 21. Coggins, J. R. , M. R. Boocock, S. Chaudhuri, J. M. Lambert, J. Lumsden, G. A. Nimmo, D. Drummond and S. Smith. 1987. The arom multifunctional enzyme from Neurospora crassa . Methods Enzymol . 142:325-341. 22. Connelly, J. A. and E. E. Conn. 1986. Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase. Z. Naturforsch. 41c : 69-78. 23. d'Amato, T. A., R. Ganson, C. G. Gaines and R. A. Jensen. 1984. Subcellular localization of chorismate- mutase isoenzymes in protoplasts from mesophyll and suspension-cultured cells of Nicotiana silvestris. Planta. 162:104-108. 24. Davis, B. J. 1967. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. 25. Deka, R. K. , C. Kleanthouse and J. R. Coggins. 1992, Identification of the essential histidine residue at the active site of Escherichia coli dehydrogenase. J. Biol. Chem. 267:22237-22242. 26. Devereux, J., Haeberli, P. and P. Marquess. 1987. The program manual for the sequence analysis software package. Genetics Computer group. Madison, WI . 27. Duncan, K. , S. Chaudhuri, M. S. Campbell and J. R. Coggins. 1986. The overexpression and complete amino acid sequence of Eschirichia coli 3-dehydroquinase. Biochem. J. 238:475-483. 28. Duncan, K. , R. M. Edwards, and J. R. Coggins. 1987. The Pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains. Biochem J. 246:375-386. 29. Dyer, W. E., L. M. Weaver, J. Zhao, D. N. Kuhn, S. C. Wellerand, K. M. Herrmann. 1990. A cDNA encoding 3-deoxy-D-arajbirjo-heptulosonate 7-phosphate synthase from Solanum tuberosum L. J. Biol. Chem. 265:1608-1614. 30. Fiedler, E., and G. Shultz . 1985. Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydroxylase from stroma of spinach chloroplasts. Plant Physiol. 79:212-218. 152 31. Gaines, C. G., G. S. Byng, R. J. Whitaker and R. A. Jensen. 1982. L-Tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris (Speg. et Comes) . Planta. 156:233-240. 32. Ganson, R. J., T. A. d'Amato and R. A. Jensen. 1986. The two- isozyme system of 3-deoxy-D-araJbino- heptulosonate 7 -phosphate synthase in Nicotiana silvestris and other higher plants. Plant Physiol. 82:203-210. 33. Gavel, Y. and G. von Heijne. 1990. A conserved cleavage- site motif in chloroplast transit peptides. FEBS . 261:455-458. 34. Geever, R. F., L. Huiet, J. A. Baum, B. M. Tyler, V. B. Patel, B. J. Rutledge, M. E. Case, and N. H. Giles. 1989. DNA sequence, organization and regulation of the qa gene cluster of Neurospora crassa. J. Mol . Biol. 207:15-34. 35. Grant, S., C. F. Roberts, H. Lamb, M. Stout and A. R. Hawkins. 1988. Genetic regulation of the quinic acid utilization (gut) gene cluster in Aspirgillus nidulans . J. Gen. Microbiol. 134:347-358. 36. Graziana, A., A. Boudet, and A. M. Boudet . 1980. Association of the quinate:NAD* oxidoreductase with one dehydroquinate hydro-lyase isoenzyme in corn seedlings. Plant 5c Cell Physio. 21:1163-1174. 37. Haslam, E., R. D. Haworth, and P. F. Knowles . 1963. The preparation and identification of 5-dehydroquinic and 5-dehydroshikimic acids. Methods Enzymol . 6:498-501. 38. Hawkins, A. R. , H. K. Lamb and C. F. Roberts. 1992. Structure of the Aspergillus nidulans gut represser- encoding gene: implications for the regulation of transcription initiation. Gene. 110:109-114. 39. Hrazdina, G. and R. A. Jensen. 1992. Spatial organization of enzymes in plant metabolic pathways. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43:241-267, 40. Jensen, R. A. 1985. The shikimate/arogenate pathway: link between carbohydrate metabolism and secondary metabolism. Physiol. Plant. 66:164-168. m 153 41. Jensen, R. A. 1986. Tyrosine and phenylalanine biosynthesis: relationship between alternative pathways, regulation and subcellular location. Rec. Adv. Phytochem. 20:57-82. 42. Jensen, R. A., L. 0. Zamir, M. St. Pierre, N. Patel and D. L. Pierson. 1977. Isolation and preparation of pretyrosine, accumulated as a dead-end metabolite by Neurospora crassa. J. Bacterid. 132:896-903. 43. Jung, E,, L. O. Zamir, and R. A. Jensen. 1986. Chloroplasts of higher plants synthesize L-phenylalanine via L-arogenate. Proc . Natl. Acad. Sci . USA. 83:7231-7235. 44. Kang, X., and R. Scheibe. 1993. Purification and characterization of the quinate :oxidoreductase from Phaseolus mungo sprouts. Phytochem. 33:769-773. 45. Keegstra, K. and L. J. Olsen. 1989. Chloroplast precursors and their transport across the envelope membranes. Annu. Rev. Plant Physiol. Plant Mol . Biol. 40:471-501. 46. Keller, B., E. Keller and F. Lingens . 1985. Arogenate dehydrogenase from Streptomyces phaeochromogenes . Biol. Chem. 366:1063-65. 47. Kleanthous, C. and J. R. Coggins . 1990. Reversible alkylation of an active site methionine residue in dehydroquinase . J. Biol. Chem. 265:10935-10939. 48. Kojima, M. , T. Minamikawa, and I. Uritani . 1969. Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing. Plant & Cell Physiol. 10:245-257. 49. Koshiba, T. 1978. Purification of two forms of the associated 3-dehydroquinate hydro- lyase and shikimate :NADP'^ oxidoreductase in Phaseolus mungo seedlings. Biochimica et Biophysica Acta. 522:10-18. 50. Koshiba, T., and S. Yoshida. 1976. Alicyclic acid metabolism in Plants 8. Association of two enzymes of the shikimate pathway in shoots of Phaseolus mungo seedlings. Plant & Cell Physiol. 17:247-253. 51. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . • :* nature. 227:680-685 r^. n 154 52. Lourenco, E. J., and V. A. Neves. 1984, Partial purification and some properties of shikimate dehydrogenase from tomatoes. Phytochemistry . 23:497-499. 53. Lourenco, E. J., G. M. Lemos Silva, and V. A. Neves. 1991. Purification and properties of shikimate dehydrogenase from cucumber {Cucumis sativus L.). J. Agric. Food Chem. 39:458-462. 54. Lutcke, H. A., K. C. Chow, F. S. Mickel, K. A. Moss, H. F. Kern and G. A. Scheele. 1987. Selection of AUG initiation codons differ in plants and animals. EMBO J. 6:43-48. 55. Margulis, L. 1970. IN: Origin of the Eukaryotic Cell. Yale Univ. Press. New Haven, Co. 56. Mayer, E., S. Waldner-Sander, B. Keller, E. Keller and F. Lingens. 1985. Purification of arogenate dehydrogenase from Phenylobacterium immobile. FEBS. 179:208-212. 57. Meredith, M. J. and R. R. Schmidt. 1991. NAD-specific glutamate dehydrogenase isoenzyme localized in mitochondria of nitrate-cultured Chlorella sorokiniana cells. Plant Physiol. (Life Sci. Adv.). 10:67-71. 58. Minamikawa, T. 1977. Quinate:NAD oxidoreductase of germinating Phaseolus mungo seeds: partial purification and some properties. Plant & Cell Physiol. 18:743-752. 59. Morris, P. F., C. A. Bonner, R. L. Doong, P. Subramaniam, R. S. Fischer, and R. A. Jensen. 1989. Mechanical wounding induces the entire primary pathway of aromatic amino acid biosynthesis. Plant Physiol. Life Sci. Adv. 8:721-733. 60. Morris, P. F. , R. L. Doong, and R. A. Jensen. 1989. Evidence from Solanum tuberosum in support of the dual- pathway hypothesis of aromatic biosynthesis. Plant Physiol. 89:10-14. 61. Mousdale, D. M., M. S. Campbell, and J. R. Coggins . 1987. Purification and characterization of bifunctional dehydroquinase-shikimate:NADP oxidoreductase from pea seedlings. Phytochemistry. 26:2665-2670. 62. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15:473-497. 155 63. Niyogi, K. K. , and G. R. Fink. 1992. Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway. The Plant Cell. 4:721-733. 64. Ogawa, K. , and T. N. Tateoka. 1988. NAD-dependent shikimate dehydrogenase from mung bean cells in suspension culture. Botanical Mag. Tokyo. 101:203-206. 65. Ossipov, V. I., and I. V. Shein. 1987. Causes of the inactivation of quinate dehydrogenase from Pinus sylvestris L. needles. Biokhimiya. 52:194. 66. Ouchterlony, 0. 1958. IN: P. Kallos (ed.). Progress and allergy. Karger, Basil. Vol V. 67. Rothe, G. M. , W. Maurer, and C. Mielke . 1976. A Study on 3-deoxy-D-araJbino-heptulosonic acid 7-phosphate synthase in higher plants. The existence of three isoenzymes in Pisum sativum. Ber. Deutsch. Bot . Ges. Bd. 89:163-173. 68. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular cloning a laboratory manual . Cold Spring Harbor Laboratory Press. Vol. 1, 2, and 3. 69. Schaller, A., J. Schmid, U. Leibinger and N. Amrhein. 1991. Molecular cloning and analysis of a cDNA coding for chorismate synthase from the higher plant Corydalis sempervirens Pers. J. Biol. Chem. 266:21434-21438. 70. Schmid, J., A. Scalier, U. Leibinger, W. Boll and N. Amrhein. 1992. The in vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts. The Plant Journal. 2:375-3 83. 71. Schmidt, C. L., D. Grundemann, G. Groth, B. Muller, H. Henning, and G. Schultz. 1991. Shikimate pathway in non-photosynthetic tissues identification of common enzymes and partial purification of dehydroquinate hydrolyase- shikimate oxidoreductase and chorismate mutase from roots. J. Plant Physiol. 138:51-56. 72. Scrutton, N. S., A. Berry and R. N. Perham. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature (London) 343:1342-1346. 156 73. Shah, D. M. , R. B. Horsch, H. J. Klee, G. M. Kishore, J. A. Winter, N. E. Turner, C. M. Hironaka, P. R. Sanders, C. S. Crasser, S. Aykent, N. R. Siegel, S. R. Rogers and R. T. Fraley. 1986. Engineering herbicide tolerance in transgenic plants. Science. 233:478-81. 74. Shaw, C. R. and R. Prasad. 1970. Starch Gel electrophoresis of enzymes. A Compilation of Recipes. Biochem. Genet. 4:297-320. 75. Siehl, D. L., and E. E. Conn. 1988. Kinetic and regulatory properties of arogenate dehydratase in seedlings of Sorghum bicolor (L.) Moench. Arch. Biochem, Biophys. 260:822-829. 76. Siehl, D. L. , B. K. Singh, and E. E. Conn. 1986. Tissue distribution and subcellular localization of prephenate aminotransferase in leaves of Sorghum bicolor. Plant Physiol. 81:711-713. 77. Smith, D. D. S., and J. R. Coggins . 1983. Isolation of a bifunctional domain from the pentafunctional arom enzyme complex of Neurospora crassa. Biochem J. 213:405-415. 78. Stafford, H. A. 1974. The metabolism of aromatic compounds. Annu. Rev. Plant Physiol. 25:459-486. 79. Weiss, U. , J. M. Edwards. 1980. In The Biosynthesis of aromatic compounds. John Wiley and Sons, New York, NY. 157 BIOGRAPHICAL SKETCH Carol A. Bonner graduated with a New York State High School Equivalency Diploma in 1967. A B.S. in biochemistry was undertaken in the Department of Biology at State University of New York at Binghamton and was completed in May of 1982. Education was continued from 1982 to 1985, whereupon an M.S. degree was completed in biological sciences in the Department of Biology at the State University of New York at Binghamton under the supervision of Professor Roy A. Jensen. At this time, the author accepted a position as Bio Science Manager at the University of Florida in the Department of Microbiology and Cell Science. A continuation of education to receive a Ph.D. degree in the Department of Microbiology and Cell Science under the supervision of Professor Roy A, Jensen was begun in the fall semester of 1989 and completed in April of 1994. 4 I certify that I have read this study and in my opinion it conforms to acceptable standards of scholarly presenta- tion and is fully adequate, in scope and quality, as a dis- sertation for the degree of Doctor of Philosophy. Roy A.*J Jensdn, Chair Professor of Microbiology and Cell Science I certify that I have read this study and in my opinion it conforms to acceptable standards of scholarly presenta- tion and is fully adequate, in scope and quality, as a dis- sertation for the degree of Doctor of Philosophy. ^. r/)A^y^^J (y- ^ Lonnie 0. Ingram Professor of Microbiology and Cell Science I certify that I have read this study and in my opinion it conforms to acceptable standards of scholarly presenta- tion and is fully adequate, in scope and quality, as a dis- sertation for the degree of Doctor of Philosophy. X J-S^Ah^^^ Schmidt Graduate Research Professor of Microbiology and Cell Science I certify that I have read this study and in my opinion it conforms to acceptable standards of scholarly presenta- tion and is fully adequate, in scope and quality, as a dis- sertation for the degree of Doctor of Philosophy. )c*i-*' Cvv-xx-iWo-. .-.^ ^ . r^vv %■ Keelnatham T. Shanmugam Professor of Microbiology and Cell Science I certify that I have read this study and in my opinion it conforms to acceptable standards of scholarly presenta- tion and is fully adequate, in scope and quality, as a dis- sertation for the d^g-cee of Doctor ,,4x£ Philosophy. Richard P. Boyce Professor of Biochemistry and Molecular Biology This dissertation was submitted to the Graduate Faculty of the College of Agriculture and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philiosophy. April 1994 Dean, 'a^A /. t^ ^ lege of Agriculture Dean, Graduate School UNIVERSITY OF FLORIDA 3 1262 08556 9753 ■f^