A monofunctional prephenate dehydrogenase created by cleavage of the 5' 109 bp of the tyrA gene from Erwinia herbicola.

A cohesive phylogenetic cluster that is limited to enteric bacteria and a few closely related genera possesses a bifunctional protein that is known as the T-protein and is encoded by tyrA. The T-protein carries catalytic domains for chorismate mutase and for cyclohexadienyl dehydrogenase. Cyclohexadienyl dehydrogenase can utilize prephenate or L-arogenate as alternative substrates. A portion of the tyr A gene cloned from Erwinia herbicola was deleted in vitro with exonuclease III and fused in-frame with a 5' portion of lacZ to yield a new gene, denoted tyrA*, in which 37 N-terminal amino acids of the T-protein are replaced by 18 amino acids encoded by the polycloning site/5' portion of the lacZ alpha-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the Km of the TyrA* dehydrogenase for NAD+ remained unaltered, the Km for prephenate was fourfold greater and the Vmax was almost twofold greater than observed for the parental T-protein dehydrogenase. Activity with L-arogenate, normally a relatively poor substrate, was reduced to a negligible level. The prephenate dehydrogenase activity encoded by tyrA* was hypersensitive to feedback inhibition by L-tyrosine (a competitive inhibitor with respect to prephenate), partly because the affinity for prephenate was reduced and partly because the Ki value for L-tyrosine was decreased from 66 microM to 14 microM. Thus, excision of a portion of the chorismate mutase domain is shown to result in multiple extra-domain effects upon the cyclohexadienyl dehydrogenase domain of the bifunctional protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The TyrA family of aromatic-pathway dehydrogenases in phylogenetic context

Background  

The TyrA protein family includes members that catalyze two dehydrogenase reactions in distinct pathways leading to L-tyrosine and a third reaction that is not part of tyrosine biosynthesis. Family members share a catalytic core region of about 30 kDa, where inhibitors operate competitively by acting as substrate mimics. This protein family typifies many that are challenging for bioinformatic analysis because of relatively modest sequence conservation and small size.     

The Crystal Structure of Aquifex aeolicus Prephenate Dehydrogenase Reveals the Mode of Tyrosine Inhibition

TyrA proteins belong to a family of dehydrogenases that are dedicated to l-tyrosine biosynthesis. The three TyrA subclasses are distinguished by their substrate specificities, namely the prephenate dehydrogenases, the arogenate dehydrogenases, and the cyclohexadienyl dehydrogenases, which utilize prephenate, l-arogenate, or both substrates, respectively. The molecular mechanism responsible for TyrA substrate selectivity and regulation is unknown. To further our understanding of TyrA-catalyzed reactions, we have determined the crystal structures of Aquifex aeolicus prephenate dehydrogenase bound with NAD⁺ plus either 4-hydroxyphenylpyuvate, 4-hydroxyphenylpropionate, or l-tyrosine and have used these structures as guides to target active site residues for site-directed mutagenesis. From a combination of mutational and structural analyses, we have demonstrated that His-147 and Arg-250 are key catalytic and binding groups, respectively, and Ser-126 participates in both catalysis and substrate binding through the ligand 4-hydroxyl group. The crystal structure revealed that tyrosine, a known inhibitor, binds directly to the active site of the enzyme and not to an allosteric site. The most interesting finding though, is that mutating His-217 relieved the inhibitory effect of tyrosine on A. aeolicus prephenate dehydrogenase. The identification of a tyrosine-insensitive mutant provides a novel avenue for designing an unregulated enzyme for application in metabolic engineering.Tyrosine serves as a precursor for the synthesis of proteins and secondary metabolites such as quinones (1-3), alkaloids (4), flavonoids (5), and phenolic compounds (5, 6). In prokaryotes and plants, these compounds are important for viability and normal development (7).The TyrA protein family consists of dehydrogenase homologues that are dedicated to the biosynthesis of l-tyrosine. These enzymes participate in two independent metabolic branches that result in the conversion of prephenate to l-tyrosine, namely the arogenate route and the 4-hydroxyphenylpyruvate (HPP)³ routes. Although both of these pathways utilize a common precursor and converge to produce a common end-product, they differ in the sequential order of enzymatic steps. Through the HPP route, prephenate is first decarboxylated by prephenate dehydrogenase (PD) to yield HPP, which is subsequently transaminated to l-tyrosine via a TyrB homologue (8). Alternatively, through the arogenate route, prephenate is first transaminated to l-arogenate by prephenate aminotransferase and then decarboxylated by arogenate dehydrogenase (AD) to yield l-tyrosine (9-11) (see Fig. 1A).Open in a separate windowFIGURE 1.A, metabolic routes from chorismate leading to the synthesis of l-tyrosine and l-phenylalanine. In the arogenate, 4-hydroxyphenylpyruvate, or phenylpyruvate route, prephenate and arogenate are branch point intermediates in both l-tyrosine and l-phenylalanine biosynthesis. Prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate with NAD⁺ to produce hydroxyphenylpyruvate, NADH, and CO₂ (40). B, a comparison of the chemical structure of the three ligands, HPP, HPpropionate, and tyrosine, used in the crystallization of A. aeolicus prephenate dehydrogenase. These ligands all have an -OH at the C4 position and a propionyl side chain at the C1 position of the ring.There are three classes of TyrA enzymes that catalyze the oxidative decarboxylation reactions in these two pathways. The enzymes are distinguished by the affinity for cyclohexadienyl substrates. PD and AD accept prephenate or l-arogenate, respectively, whereas the cyclohexadienyl dehydrogenases can catalyze the reaction using either substrate (12).To ensure efficient metabolite distribution of the pathway intermediates, TyrA enzymes are highly regulated by various control mechanisms, including feedback inhibition, and genetic regulation by the Tyr operon (13-16). In some cases, l-tyrosine competes directly with substrate, be it prephenate or l-arogenate for the active site of arogenate or cyclohexadienyl dehydrogenases (14, 17-19). The product HPP can also serve as an efficient competitive inhibitor with respect to prephenate (20). Additionally, at the protein level PDs are only shown to be regulated at distinct allosteric sites or domains to modulate their activity. For example, the results of kinetic studies on the bifunctional Escherichia coli chorismate mutase-prephenate dehydrogenase (CM-PD) have indicated that this enzyme likely possesses a distinct allosteric site for binding tyrosine (21). In contrast, the Bacillus subtilis PD is the only enzyme reported to be competitively inhibited by HPP and l-tyrosine but is also noncompetitively inhibited by l-phenylalanine and l-tryptophan (12, 22). Additional regulatory control is thought to originate through a C-terminal aspartate kinase-CM-TyrA domain of the B. subtilis PD (23).Biochemical analyses of PD from E. coli CM-PD have provided a framework for understanding the molecular mechanism of the TyrA enzymes. The E. coli PD-catalyzed reaction proceeds though a rapid equilibrium, random kinetic mechanism with catalysis as the rate-limiting step (24). Additionally, studies of the pH dependence of the kinetic parameters V and V/K indicate that a deprotonated group facilitates hydride transfer from prephenate to NAD⁺ by polarizing the 4-hydroxyl group of prephenate, whereas a protonated residue is required for binding prephenate to the enzyme·NAD⁺ complex (25). The conserved residues His-197 and Arg-294 have been identified through extensive mutagenesis studies to fulfill these two roles (26, 27). Further analyses of the activities of wild-type protein and site-directed variants in the presence of a series of inhibitory substrate analogues support the idea that Arg-294 binds prephenate through the ring carboxylate (26).The structures of AD from Synechocystis sp. and PD from Aquifex aeolicus (both in complex with NAD⁺) have been reported by Legrand et al. (28) and by our group (29), respectively. Analyses of these structures have provided structural information on the conserved histidine and arginine residues. The structure A. aeolicus PD has also led to the identification of other active site residues that may play a role in enzyme catalysis, most notably Ser-126, which we propose facilitates catalysis by orienting the catalytic histidine and the nicotinamide moiety of NAD⁺ into their catalytically efficient conformations. Ambiguities can arise from examination of the binary complexes, because prephenate has only been modeled in the active site. For example, analysis of the AD structure by Legrand et al. (28) places Arg-217 (equivalent to Arg-294 in E. coli and Arg-250 in A. aeolicus) too far from the active site to play a role in prephenate binding. Thus, the full complement of interactions between prephenate and TyrA proteins are still largely unknown, as are the interactions of the enzymes with l-tyrosine.To further investigate the importance of residues involved in ligand binding, specificity, and catalysis, we have carried out co-crystallization studies of A. aeolicus PD with NAD⁺ and prephenate, with NAD⁺ and 4-hydroxyphenylpropionate (HPpropionate), a product analogue, and with NAD⁺ and l-tyrosine. Accordingly, this study provides the first direct evidence that l-tyrosine binds to the active site of a prephenate dehydrogenase. We have investigated the role of Ser-126, His-147, His-217, and Arg-250 through the kinetic analysis of site-directed mutants and structural analysis of the co-crystal complexes. To understand the role of active site residues in substrate selectivity, comparative structural analysis of AD and PD was also conducted. The current study provides a basis for understanding the mechanism of substrate selectivity between the different classes of TyrA enzymes and details how A. aeolicus PD can accept prephenate as substrate and l-tyrosine as a competitive inhibitor.     

Identification of a metagenome-derived prephenate dehydrogenase gene from an alkaline-polluted soil microorganism

A novel prephenate dehydrogenase gene designated pdhE-1 was cloned by sequence-based screening of a plasmid metagenomic library from uncultured alkaline-polluted microorganisms. The deduced amino acid sequence comparison and phylogenetic analysis indicated that PdhE-1 and other putative prephenate dehydrogenases were closely related. The putative prephenate dehydrogenase gene was subcloned into pETBlue-2 vector and overexpressed in Escherichia coli BL21(DE3) pLacI. The recombinant protein was purified to homogeneity. The maximum activity of the PdhE-1 protein occurred at pH 8.0 and 45 °C using prephenic acid as the substrate. The prephenate dehydrogenase had an apparent K _m value of 0.87 mM, a V _max value of 41.5 U/mg, a k _cat value of 604.8/min and a k _cat/K _m value of 1.16 × 10⁴/mol/s. l-Tyrosine did not obviously inhibit the recombinant PdhE-1 protein. The identification of a metagnome-derived prephenate dehydrogenase provides novel material for studies and application of proteins involved in tyrosine biosynthesis.     

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.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

Construction of a heat-inducible Escherichia coli strain for efficient de novo biosynthesis of l-tyrosine

l-Tyrosine is an important amino acid widely used in food, agriculture, and pharmaceutical industries. However, the industrial application was severely constrained due to low production. To obtain the Escherichia coli mutant producing l-tyrosine in abundance, the heat-inducible expression vector carrying the two feedback resistance enzymes (3-deoxy-7-phosphoheptulonate synthase encoded by aroG^fbr and chorismate mutase/prephenate dehydrogenase encoded by tyrA^fbr) were introduced into the phenylalanine-producing E. coli strain to enable it to synthesize l-tyrosine directly from glucose. Furthermore, the CRISPR-Cas9 technology was applied to eliminate l-phenylalanine and l-tryptophan pathways for their competition for the carbon flux. The global regulatory protein TyrR, which mediates the biosynthesis and transportation of aromatic amino acids, was also deleted to increase l-tyrosine production. Among the recombinant strains, the pheA/tyrR double-gene deletion strain had the highest yield of 5.84 g/L on shake flasks. The feeding strategies were then optimized in a 3-L fermentor. The pheA/tyrR double-gene deletion strain with the heat-inducible expression plasmid pAP-aroG^fbr-tyrA^fbr was able to produce 55.54 g/L l-tyrosine by fed-batch fermentation; the substrate conversion rate was 0.25 g/g. The recombinant strains constructed in this study could be an industrial platform for the microbial production of l-tyrosine directly from glucose.     

Enzymology of l-Tyrosine Biosynthesis in Mung Bean (Vigna radiata [L.] Wilczek)

The enzymes of the 4-hydroxyphenylpyruvate (prephenate dehydrogenase and 4-hydroxyphenylpyruvate aminotransferase) and pretyrosine (prephenate aminotransferase and pretyrosine dehydrogenase) pathways of l-tyrosine biosynthesis were partially purified from mung bean (Vigna radiata [L.] Wilczek) seedlings. NADP-dependent prephenate dehydrogenase and pretyrosine dehydrogenase activities coeluted from ion exchange, adsorption, and gel-filtration columns, suggesting that a single protein (52,000 daltons) catalyzes both reactions. The ratio of the activities of partially purified prephenate to pretyrosine dehydrogenase was constant during all purification steps as well as after partial inactivation caused by p-hydroxymercuribenzoic acid or heat. The activity of prephenate dehydrogenase, but not of pretyrosine dehydrogenase, was inhibited by l-tyrosine at nonsaturating levels of substrate. The K(m) values for prephenate and pretyrosine were similar, but the specific activity with prephenate was 2.9 times greater than with pretyrosine.Two peaks of aromatic aminotransferase activity utilizing l-glutamate or l-aspartate as amino donors and 4-hydroxyphenylpyruvate, phenylpyruvate, and/or prephenate as keto acid substrates were eluted from DEAE-cellulose. Of the three keto acid substrates, 4-hydroxyphenylpyruvate was preferentially utilized by 4-hydroxyphenylpyruvate aminotransferase whereas prephenate was best utilized by prephenate aminotransferase. The identity of a product of prephenate aminotransferase as pretyrosine following reaction with prephenate was established by thin layer chromatography of the dansyl-derivative.     

A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level

The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca²⁺-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca²⁺-dependent conformational change. The Ca²⁺-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca²⁺-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca²⁺ conditions. Because TyrA activity exhibits robust changes in response to Ca²⁺-dependent-EF-hand conformational alterations, TyrA could be a good Ca²⁺-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca²⁺-dependent conformational change.     

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.     

Construction of an l-phenylalanine-producing tyrosine-prototrophic Escherichia coli strain using tyrA ssrA-like tagged alleles

To construct a Phe-producing Tyr⁺ Escherichia coli strain, TyrA (chorismate mutase/prephenate dehydrogenase) activity was varied by engineering a proteolytically unstable protein. The tyrA in the E. coli BW25113 was altered to include ssrA-like tags. The tagged tyrA genes, which ensured different growth rates in M9 medium, were introduced into a Phe-producing strain to replace ΔtyrA. Strains with unstable TyrA-(A)ANDENYALAA proteins had a lower biomass yield and a higher Phe accumulation than strains generating the more stable TyrA-(A)ANDENYALDD. The Tyr/Phe ratio produced by the TyrA-tag strains was 10-fold less than that produced by the TyrA^wt strain.     

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD⁺ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high K_m values of PDH from Streptococcus mutans.     

The prephenate dehydrogenase component of the bifunctional T-protein in enteric bacteria can utilize L-arogenate

The prephenate dehydrogenase component of the bifunctional T-protein (chorismate mutase:prephenate dehydrogenase) has been shown to utilize L-arogenate, a common precursor of phenylalanine and tyrosine in nature, as a substrate. Partially purified T-protein from Klebsiella pneumoniae and from Escherichia coli strains K 12, B, C and W was used to demonstrate the utilization of L-arogenate as an alternative substrate for prephenate in the presence of nicotinamide adenine dinucleotide as cofactor. The formation of L-tyrosine from L-arogenate by the T-protein dehydrogenase was confirmed by high-performance liquid chromatography. As expected of a common catalytic site, dehydrogenase activity with either prephenate or L-arogenate was highly sensitive to inhibition by L-tyrosine.     

Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.     

A mechanistic and electrochemical study of the interaction between dimethyl sulfide dehydrogenase and its electron transfer partner cytochrome c 2

Dimethyl sulfide dehydrogenase isolated from the photosynthetic bacterium Rhodovulum sulfidophilum is a heterotrimeric enzyme containing a molybdenum cofactor at its catalytic site, as well as five iron–sulfur clusters and a heme b cofactor. It oxidizes dimethyl sulfide (DMS) to dimethyl sulfoxide in its native role and transfers electrons to the photochemical reaction center. There is genetic evidence that cytochrome c ₂ mediates this process, and the steady state kinetics experiments reported here demonstrated that cytochrome c ₂ accepts electrons from DMS dehydrogenase. At saturating concentrations of both substrate (DMS) and cosubstrate (cytochrome c ₂), Michaelis constants, K _M,DMS and K _M,cyt of 53 and 21 μM, respectively, were determined at pH 8. Further kinetic analysis revealed a “ping-pong” enzyme reaction mechanism for DMS dehydrogenase with its two reactants. Direct cyclic voltammetry of cytochrome c ₂ immobilized within a polymer film cast on a glassy carbon electrode revealed a reversible Fe^III/II couple at +328 mV versus the normal hydrogen electrode at pH 8. The Fe^III/II redox potential exhibited only minor pH dependence. In the presence of DMS dehydrogenase and DMS, the peak-shaped voltammogram of cytochrome c ₂ is transformed into a sigmoidal curve consistent with a steady-state (catalytic) reaction. The cytochrome c ₂ effectively mediates electron transfer between the electrode and DMS dehydrogenase during turnover and a significantly lower apparent electrochemical Michaelis constant of 13(±1) μM was obtained. The pH optimum for catalytic DMS oxidation by DMS dehydrogenase with cytochrome c ₂ as the electron acceptor was found to be approximately 8.3.     

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.     

The organisation of methanol dehydrogenase and c-type cytochromes on the respiratory membrane of Methylophilus methylotrophus

The membrane-impermeant, protein-labelling reagent [¹⁴C]isethionyl acetimidate has been used to investigate the detailed topography of the membrane-associated methanol oxidase system in the methylotrophic bacterium Methylophilus methylotrophus. The results show that methanol dehydrogenase and cytochrome c₁ are significantly less exposed on the periplasmic surface of the membrane than either cytochrome C_H or an as yet unidentified 12-kDa protein. The partial crypticity of the methanol dehydrogenase has been confirmed by fingerprinting with V8 protease. The results are discussed in terms of the electron transfer functions of the various redox proteins.     

Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism

The enzyme prephenate dehydrogenase catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate for the biosynthesis of tyrosine. Prephenate dehydrogenases exist as either monofunctional or bifunctional enzymes. The bifunctional enzymes are diverse, since the prephenate dehydrogenase domain is associated with other enzymes, such as chorismate mutase and 3-phosphoskimate 1-carboxyvinyltransferase. We report the first crystal structure of a monofunctional prephenate dehydrogenase enzyme from the hyper-thermophile Aquifex aeolicus in complex with NAD+. This protein consists of two structural domains, a modified nucleotide-binding domain and a novel helical prephenate binding domain. The active site of prephenate dehydrogenase is formed at the domain interface and is shared between the subunits of the dimer. We infer from the structure that access to the active site is regulated via a gated mechanism, which is modulated by an ionic network involving a conserved arginine, Arg250. In addition, the crystal structure reveals for the first time the positions of a number of key catalytic residues and the identity of other active site residues that may participate in the reaction mechanism; these residues include Ser126 and Lys246 and the catalytic histidine, His147. Analysis of the structure further reveals that two secondary structure elements, beta3 and beta7, are missing in the prephenate dehydrogenase domain of the bifunctional chorismate mutase-prephenate dehydrogenase enzymes. This observation suggests that the two functional domains of chorismate mutase-prephenate dehydrogenase are interdependent and explains why these domains cannot be separated.     

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD+

A new, acyclic NAD-analog, acycloNAD⁺ has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD⁺ with a redox potential of −324 mV and a 341 nm λ_max for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD⁺ by primary alcohols. With HLADH 1-butanol has the highest V_max at 49% that of β-NAD⁺. The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon–hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD⁺. In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD⁺ by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD⁺ has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases.     

Purification and characterization of glucose-6-phosphate dehydrogenase from Aspergillus parasiticus

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was purified from mycelium of Aspergillus parasiticus (1-11-105 Whl). The enzyme had a molecular weight of 1.8 × 10⁵ and was composed of four subunits of apparently equal size. The substrate specificity was very strict, only glucose 6-phosphate and glucose being oxidized by NADP or thio-NADP. Zinc ion was a powerful inhibitor of the enzyme, inhibition being competitive with respect to glucose 6-phosphate, with K_i about 2.5 μm. Other divalent metal ions which also serve as inhibitors are nickel, cadmium, and cobalt. It is proposed that the stimulation of polyketide synthesis by zinc ion may be mediated in part by inhibition of glucose-6-phosphate dehydrogenase.