Arogenate dehydrogenase from Streptomyces phaeochromogenes. Purification and properties

Arogenate dehydrogenase, the terminal enzyme of tyrosine biosynthesis in Streptomyces phaeochromogenes, was purified to homogeneity by a five-step procedure. The enzyme is a dimer of Mr 57 600 as determined by dodecyl sulfate polyacrylamide gel electrophoresis after cross-linking of the monomers, or of 66 300 as found by gel permeation chromatography, and consists of two identical subunits of Mr 28 100. The pI of the enzyme is 4.45, and the Km values are 0.105mM for arogenate and 0.01 mM for NAD.     

A single cyclohexadienyl dehydrogenase specifies the prephenate dehydrogenase and arogenate dehydrogenase components of the dual pathways to L-tyrosine in Pseudomonas aeruginosa

Dual biosynthetic pathways diverge from prephenate to L-tyrosine in Pseudomonas aeruginosa, with 4-hydroxyphenylpyruvate and L-arogenate being the unique intermediates of these pathways. Prephenate dehydrogenase and arogenate dehydrogenase activities could not be separated throughout fractionation steps yielding a purification of more than 200-fold, and the ratio of activities was constant throughout purification. Thus, the enzyme is a cyclohexadienyl dehydrogenase. The native enzyme has a molecular weight of 150,000 and is a hexamer made up of identical 25,500 subunits. The enzyme is specific for NAD+ as an electron acceptor, and identical Km values of 0.25 mM were obtained for NAD+, regardless of whether activity was assayed as prephenate dehydrogenase or as arogenate dehydrogenase. Km values of 0.07 mM and 0.17 mM were calculated for prephenate and L-arogenate, respectively. Inhibition by L-tyrosine was noncompetitive with respect to NAD+, but was strictly competitive with respect to either prephenate or L-arogenate. With cyclohexadiene as variable substrate, similar Ki values for L-tyrosine of 0.06 mM (prephenate) and 0.05 mM (L-arogenate) were obtained. With NAD+ as the variable substrate, similar Ki values for L-tyrosine of 0.26 mM (prephenate) and 0.28 mM (L-arogenate), respectively, were calculated. This is the first characterization of a purified, monofunctional cyclohexadienyl dehydrogenase.     

Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana.

The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity. Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2. Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not. The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady-state kinetics. For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E-NADPH-arogenate and E-NADP-tyrosine, are formed.     

A single cyclohexadienyl dehydratase specifies the prephenate dehydratase and arogenate dehydratase components of one of two independent pathways to L-phenylalanine in Erwinia herbicola

Dual biosynthetic pathways diverge from prephenate to L-phenylalanine in Erwinia herbicola, the unique intermediates of these pathways being phenylpyruvate and L-arogenate. After separation from the bifunctional P-protein (one component of which has prephenate dehydratase activity), the remaining prephenate dehydratase activity could not be separated from arogenate dehydratase activity throughout fractionation steps yielding a purification of more than 1200-fold. The ratio of activities was constant after removal of the P-protein, and the two dehydratase activities were stable during purification. Hence, the enzyme is a cyclohexadienyl dehydratase. The native enzyme has a molecular mass of 73 kDa and is a tetramer made up of identical 18-kDa subunits. Km values of 0.17 mM and 0.09 mM were calculated for prephenate and L-arogenate, respectively. L-Arogenate inhibited prephenate dehydratase competitively with respect to prephenate, whereas prephenate inhibited arogenate dehydratase competitively with respect to L-arogenate. Thus, the enzyme has a common catalytic site for utilization of prephenate or L-arogenate as alternative substrates. This is the first characterization of a purified monofunctional cyclohexadienyl dehydratase.     

L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes

The biosynthetic route to L-tyrosine was identified in isogenic suspension-cultured cells of N. silvestris. Arogenate (NADP⁺) dehydrogenase, the essential enzyme responsible for the conversion of L-arogenato L-tyrosine, was readily observed in crude extracts. In contrast, prephenate dehydrogenase (EC 1.3.1.13) activity with either NAD⁺ or NADP⁺ was absent altogether. Therefore, it seems likely that this tobacco species utilizes the arogenate pathway as the exclusive metabolic route to L-tyrosine. L-Tyrosine (but not L-phenylalanine) was a very effective endproduct inhibitor of arogenate dehydrogenase. In addition, analogs of L-tyrosine (m-fluoro-DL-tyrosine [MFT], D-tyrosine and N-acetyl-DL-tyrosine), but not of L-phenylalanine (o-fluoro-DL-phenylalanine and p-fluoro-DL-phenylalanine), were able to cause inhibition of arogenate dehydrogenase. The potent antimetabolite of L-tryptophan, 6-fluoro-DL-tryptophan, had no effect upon arogenate dehydrogenase activity. Of the compounds tested, MFT was actually more effective as an inhibitor of arogenate dehydrogenase than was L-tyrosine. Since MFT was found to be a potent antimetabolite inhibitor of growth in N. silvestris and since inhibition was specifically and effectively reversed by L-tyrosine, arogenate dehydrogenase is an outstanding candidate as the in vivo target of analog action. Although chorismate mutase (EC 5.4.99.5) cannot be the prime target of MFT action, MFT can mimick L-tyrosine in partially inhibiting this enzyme activity. The activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was insensitive to L-phenylalanine or L-tyrosine. The overall features of this system indicate that MFT should be a very effective analog mimick for selection of feedback-insensitive regulatory mutants L-tyrosine biosynthesis.Abbreviations DAHP synthase 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase - 6FT 6-fluoro-DL-tryptophan - MFT m-fluoro-DL-tyrosine - OFP o-fluoro-DL-phenylalanine - PFP p-fluoro-DL-phenylalanine     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

Clues from Xanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

The recent placement of major Gram-negative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading to Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping, Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping. X. campestris is like P. aeruginosa (and unlike E. coli) in its possession of dual flow routes to both L-phenylalanine and L-tyrosine from prephenate. Like all other members of Superfamily B, X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that of P. aeruginosa in X. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-D-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., in E. coli), as two isozymes (e.g., in P. aeruginosa), or as one enzyme (in X. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus, E. coli has gained an isozyme, whereas X. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme of X. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. In X. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway in X. campestris is thus far unique in nature.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.     

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.     

Purification and properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Biochemical diversity for biosynthesis of aromatic amino acids among the cyanobacteria.

We examined the enzymology and regulatory patterns of the aromatic amino acid pathway in 48 strains of cyanobacteria including representatives from each of the five major grouping. Extensive diversity was found in allosteric inhibition patterns of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, not only between the major groupings but also within several of the generic groupings. Unimetabolite inhibition by phenylalanine occurred in approximately half of the strains examined; in the other strains unimetabolite inhibition by tyrosine and cumulative, concerted, and additive patterns were found. The additive patterns suggest the presence of regulatory isozymes. Even though both arogenate and prephenate dehydrogenase activities were found in some strains, it seems clear that the arogenate pathway to tyrosine is a common trait that has been highly conserved among cyanobacteria. No arogenate dehydratase activities were found. In general, prephenate dehydratase activities were activated by tyrosine and inhibited by phenylalanine. Chorismate mutase, arogenate dehydrogenase, and shikimate dehydrogenase were nearly always unregulated. Most strains preferred NADP as the cofactor for the dehydrogenase activities. The diversity in the allosteric inhibition patterns for 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase, cofactor specificities, and the presence or absence of prephenate dehydrogenase activity allowed the separation of subgroupings within several of the form genera, namely, Synechococcus, Synechocystis, Anabaena, Nostoc, and Calothrix.     

Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction

The extreme diversity in substrate specificity, and in the regulation mechanism of arogenate/prephenate dehydrogenase enzymes in nature, makes a comparative structural study of these enzymes of great interest. We report here on the biochemical and structural characterization of arogenate dehydrogenase from Synechocystis sp. (TyrAsy). This work paves the way for the understanding of the structural determinants leading to diversity in substrate specificity, and of the regulation mechanisms of arogenate/prephenate dehydrogenases. The overall structure of TyrAsy in complex with NADP was refined to 1.6 A. The asymmetric unit contains two TyrAsy homodimers, with each monomer consisting of a nucleotide binding N-terminal domain and a particularly unique alpha-helical C-terminal dimerization domain. The substrate arogenate was modeled into the active site. The model of the ternary complex enzyme-NADP-arogenate nicely reveals at the atomic level the concerted mechanism of the arogenate/prephenate dehydrogenase reaction.     

Terminal phenylalanine and tyrosine biosynthesis of Microtetraspora glauca

The enzymes of the terminal steps of the phenylalanine and tyrosine biosynthesis were partially purified and characterized in Microtetraspora glauca, a spore-forming member of the order Actinomycetales. This bacterium relies exclusively on the phenylpyruvate route for phenylalanine synthesis, no arogenate dehydratase activity being found. Prephenate dehydratase is subject to feedback inhibition by phenylalanine, tyrosine and tryptophan, each acting as competitive inhibitor by increasing the Km of 72 microM for prephenate. Based on the results of gel chromatography on Sephadex G-200, the molecular mass of about 110,000 Da is not altered by any of the effectors. The enzyme is quite sensitive to inhibition by 4-hydroxymercuribenzoate. Microtetraspora glauca can utilize arogenate and 4-hydroxyphenylpyruvate as intermediates in tyrosine biosynthesis. Prephenate and arogenate dehydrogenase activities copurifying from ion exchange columns with coincident profiles were detected. From gel-filtration columns the two activities eluted at an identical molecular-mass position of about 68,000 Da. The existence of a single protein exhibiting substrate ambiguity is consistent with the findings, that both dehydrogenases have similar chromatographic properties, exhibit cofactor requirement for NAD and are inhibited to the same extent by tyrosine and 4-hydroxymercuribenzoate.     

Synthesis of chiral arogenic acid via immobilized microbial proteins

Although l-(8S)-arogenate has been recognized as a potential precursor of l-phenylalanine or l-tyrosine biosynthesis for only a few years, it is widely distributed in nature. The biochemical formation of arogenate has involved its isolation from the culture supernatant of a mutant strain of Neurospora crassa, a lengthy procedure of 20-day duration. We now report an improved approach using immobilized crude enzyme extracts from a cyanobacterium. The starting materials, chorismic acid or prephenic acid, are readily available, and overall yields ranging from 40 to 60% are obtained. The whole procedure takes only 1 day. Crude, unfractionated enzyme extracts from Synechocystis sp. ATCC 29108 are immobilized on a phenoxyacetyl cellulose solid support. The hydrophobic binding of the extract proteins did not denature chorismate mutase or prephenate aminotransferase, the enzymes catalyzing the conversion of chorismate to prephenate and prephenate to arogenate, respectively. This microbial system was ideally suited for preparation of arogenate, since other enzyme activities which might compete for prephenate or chorismate as substrates, or which might further metabolize arogenate, were absent or inactive under the conditions used. In addition to the substrates prephenate or chorismate, pyridoxal-5′-phosphate (the coenzyme required for transamination), as well as leucine (amino donor for transamination of prephenate), was added. The reaction product, arogenate, was separated from the starting materials by preparative thin-layer chromatography.     

Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii.

Formaldehyde hydrogenase and formate dehydrogenase were purified 130-fold and 19-fold respectively from Candida boidinii grown on methanol. The final enzyme preparations were homogenous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weights of the enzymes were determined by sedimentation equilibrium studies and calculated as 80000 and 74000 respectively. Dissociation into subunits was observed by treatment with sodium dodecylsulfate. The molecular weights of the polypeptide chains were estimated to be 40000 and 36000 respectively. The NAD-linked formaldehyde dehydrogenase specifically requires reduced glutathione for activity. Besides formaldehyde only methylglyoxal served as a substrate but no other aldehyde tested. The Km values were found to be 0.25 mM for formaldehyde, 1.2 mM for methylglyoxal, 0.09 mM for NAD and 0.13 mM for glutathione. Evidence is presented which demonstrates that the reaction product of the formaldehyde-dehydrogenase-catalyzed oxidation of formaldehyde is S-formylglutathione rather than formate. The NAD-linked formate dehydrogenase catalyzes specifically the oxidation of formate to carbon dioxide. The Km values were found to be 13 mM for formate and 0.09 mM for NAD.     

Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli.

Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme.     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

Clues fromXanthomonas campestris about the evolution of aromatic biosynthesis and its regulation

Summary The recent placement of major Gramnegative prokaryotes (Superfamily B) on a phylogenetic tree (including, e.g., lineages leading toEscherichia coli, Pseudomonas aeruginosa, andAcinetobacter calcoaceticus) has allowed initial insights into the evolution of the biochemical pathway for aromatic amino acid biosynthesis and its regulation to be obtained. Within this prokaryote grouping,Xanthomonas campestris ATCC 12612 (a representative of the Group V pseudomonads) has played a key role in facilitating deductions about the major evolutionary events that shaped the character of aromatic biosynthesis within this grouping.X. campestris is likeP. aeruginosa (and unlikeE. coli) in its possession of dual flow routes to bothl-phenylalanine andl-tyrosine from prephenate. Like all other members of Superfamily B,X. campestris possesses a bifunctional P-protein bearing the activities of both chorismate mutase and prephenate dehydratase. We have found an unregulated arogenate dehydratase similar to that ofP. aeruginosa inX. campestris. We separated the two tyrosine-branch dehydrogenase activities (prephenate dehydrogenase and arogenate dehydrogenase); this marks the first time this has been accomplished in an organism in which these two activities coexist. Superfamily B organisms possess 3-deoxy-d-arabino-heptulosonate 7-P (DAHP) synthase as three isozymes (e.g., inE. coli), as two isozymes (e.g., inP. aeruginosa), or as one enzyme (inX. campestris). The two-isozyme system has been deduced to correspond to the ancestral state of Superfamily B. Thus,E. coli has gained an isozyme, whereasX. campestris has lost one. We conclude that the single, chorismate-sensitive DAHP synthase enzyme ofX. campestris is evolutionarily related to the tryptophan-sensitive DAHP synthase present throughout the rest of Superfamily B. InX. campestris, arogenate dehydrogenase, prephenate dehydrogenase, the P-protein, chorismate mutase-F, anthranilate synthase, and DAHP synthase are all allosteric proteins; we compared their regulatory properties with those of enzymes of other Superfamily B members with respect to the evolution of regulatory properties. The network of sequentially operating circuits of allosteric control that exists for feedback regulation of overall carbon flow through the aromatic pathway inX. campestris is thus far unique in nature.