Use of Site-Directed Mutagenesis To Identify Residues Specific for Each Reaction Catalyzed by Chorismate Mutase−Prephenate Dehydrogenase from <i>Escherichia coli</i>

Christendat, Dinesh; Saridakis, V.; Turnbull, Joanne L.

doi:10.1021/bi981412b

Cited by 28 publications

(44 citation statements)

References 54 publications

(121 reference statements)

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“…Detailed kinetic and pH rate analysis for AroE shikimate dehydrogenase from the pea also displayed a similar pH rate profile (24). Active site lysine and histidine residues are known to titrate in this region (range pH 6.5 to 8.5); however, other active site groups, such as aspartate, can also titrate in this pH range depending on their environment (25). This observation is in agreement with the crystal structure of HI0607, which showed that Lys-67 and Asp-103 are located at the entrance of the substrate-binding pocket, the expected position for a catalytic group in this protein (Fig.…”

Section: Identification Of the Nucleotide And Substrate-binding Domains-mentioning

confidence: 80%

Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

Singh

Korolev

Королева

et al. 2005

Journal of Biological Chemistry

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To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ϳ1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase.The shikimate pathway occupies a central position for aromatic biosynthesis in microbes and plants but is not present in humans and other higher animals. The absence of the shikimate pathway in animals makes it an ideal target for herbicide and anti-microbial drug design. Recently the shikimate pathway was identified in apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, which has renewed interest in better understanding the enzymes in the pathway (1, 2). The importance of the shikimate pathway is exemplified by the common herbicide glyphosate, which inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Recent studies have also shown that glyphosate blocks the shikimate pathway in apicomplexan parasites and is effective in controlling their growth (3). The shikimate pathway consists of seven enzymatic steps initiated by the condensation of phosphoenolpyruvate and erythrose-4-phosphate by 3-deoxy-Darabino-heptolosonate 7-phosphate synthase. The last enzymatic step produces the branch point intermediate, chorismate, which serves in turn as the precursor for a number of pathways including those involved in aromatic amino acid, phytoalexin, flavanoid, and lignin biosynthesis.The fourth enzyme in the pathway, shikimate dehydrogenase (shikimate:NADP ϩ oxidoreductase; EC 1.1.1.25), is involved in the NADPH-dependent reduction of dehydroshikimate to shikimate. This enzymatic reaction proceeds in both the forward and reverse direction with similar rates and similar Michaelis constants for substrates in either direction. Kinetic studies with substrate analogues and isotope exchange demonstrated that the shikimate dehydrogena...

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Section: Identification Of the Nucleotide And Substrate-binding Domains-mentioning

confidence: 80%

Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

Singh

Korolev

Королева

et al. 2005

Journal of Biological Chemistry

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“…An earlier report by Christendat et al [15] indicated that mutagenesis of several residues in the dehydrogenase portion of the T-protein significantly affected CM activity, either by reducing K cat (His189Asn) or elevating K m (His239Asn, His245Asn). The findings reported here suggest that additional amino acids in the PDH domain, extending beyond residue 336 effect mutase activity.…”

Section: Discussionmentioning

confidence: 99%

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T‐protein

Chen

Vincent

Wilson³

et al. 2003

European Journal of Biochemistry

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The Escherichia coli bifunctional T-protein transforms chorismic acid to p-hydroxyphenylpyruvic acid in the L-tyrosine biosynthetic pathway. The 373 amino acid T-protein is a homodimer that exhibits chorismate mutase (CM) and prephenate dehydrogenase (PDH) activities, both of which are feedback-inhibited by tyrosine. Fifteen genes coding for the T-protein and various fragments thereof were constructed and successfully expressed in order to characterize the CM, PDH and regulatory domains. Residues 1-88 constituted a functional CM domain, which was also dimeric. Both the PDH and the feedback-inhibition activities were localized in residues 94-373, but could not be separated into discrete domains. The activities of cloned CM and PDH domains were comparatively low, suggesting some cooperative interactions in the native state. Activity data further indicate that the PDH domain, in which NAD, prephenate and tyrosine binding sites were present, was more unstable than the CM domain.

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“…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 ϩ by polarizing the 4-hydroxyl group of prephenate, whereas a protonated residue is required for binding prephenate to the enzyme⅐NAD ϩ 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).…”

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confidence: 99%

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

Sun

Shahinas

Bonvin

et al. 2009

Journal of Biological Chemistry

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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).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, T...

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Use of Site-Directed Mutagenesis To Identify Residues Specific for Each Reaction Catalyzed by Chorismate Mutase−Prephenate Dehydrogenase from Escherichia coli

Cited by 28 publications

References 54 publications

Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

Mapping of chorismate mutase and prephenate dehydrogenase domains in the Escherichia coli T‐protein

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

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