Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product

Maruya, Aiko; O'Connor, Mary Jane; Backman, Keith

doi:10.1128/jb.169.10.4852-4853.1987

Cited by 24 publications

(12 citation statements)

References 15 publications

(13 reference statements)

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“…Most mechanistic studies on the enzymic transformation have used the bifunctional "T" protein from Escherichia coli, in which the activity of chorismate mutase and that of prephenate dehydrogenase lie in different active sites on the same polypeptide chain (Koch et al, 1971). The mutase activity is thought to derive from the N-terminal third of the protein (Hudson & Davidson, 1984;Maruya et al, 1987). Both the enzymic and the nonenzymic reactions proceed via transition states of chairlike geometry (Sogo et al, 1984;Copley & Knowles, 1985).…”

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confidence: 99%

Monofunctional Chorismate Mutase from Bacillus subtilis: FTIR Studies and the Mechanism of Action of the Enzyme

Gray¹,

Knowles²

1994

Biochemistry

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The Fourier transform infrared (FTIR) spectrum of the complex between prephenate and the monofunctional chorismate mutase from Bacillus subtilis displays one prominent band at 1714 cm-1. Using isotopically-labeled ligand, we have shown that this band corresponds to the ketonic carbonyl stretching vibration of enzyme-bound prephenate. The frequency of this carbonyl vibration of prephenate does not change significantly on binding to the protein. These data indicate that chorismate mutase does not use electrophilic catalysis in the rearrangement of chorismate. A comparison of the resolution-enhanced FTIR spectra of the unliganded mutase and of the protein complexed with its ligands reveals marked differences in the amide I' vibration band. These changes suggest that structural alterations in the protein occur upon binding prephenate. When combined with information from the crystal structure of the enzyme and its complexes, it appears that significant ordering of the C-terminal region occurs upon ligand binding. These changes at the active site may be important for efficient catalysis and likely influence the association and dissociation rates of the enzyme and its ligands. The enzymic rearrangement of chorismate evidently proceeds via a pericyclic process, and much, if not all, of the rate acceleration derives from the selective binding of the appropriate conformer of the substrate, with some additional contribution possible from electrostatic stabilization of the transition state.

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Monofunctional Chorismate Mutase from Bacillus subtilis: FTIR Studies and the Mechanism of Action of the Enzyme

Gray¹,

Knowles²

1994

Biochemistry

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“…A genetic construct derived from pheA has also been described (Stewart et al, 1990) which encodes a monofunctional CM-P that lacks the prephenate dehydratase domain. The separability of the two T-protein functions had remained in doubt (Rood et al, 1982) until recently, when molecular-genetic approaches established the genetic separability of functional CM-T and dehydrogenase components in E. coli (Maruya et al, 1987). In the latter study, no enzymological characterizations of the new monofunctional enzymes were given.…”

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confidence: 99%

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

Xia

Zhao²,

Fischer³

et al. 1992

Journal of General Microbiology

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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 tyrA gene cloned from Erwinia herbicola was deleted in vifru with exonuclease I11 and fused in-frame with a 5' portion of lac2 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/S portion of the lac2 a-peptide of pUC19. The TyrA* protein retained dehydrogenase activity but lacked mutase activity, thus demonstrating the separability of the two catalytic domains. While the K, of the TyrA* dehydrogenase for NAD+ remained unaltered, the K, 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 VM to 14 PM. 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. These include alterations in apparent substrate specificity, isoelectric point, stability, catalytic properties and regulatory properties.

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“…investigated organisms such as Escherichia coli employ two bifunctional enzymes: a chorismate mutase-prephenate de-hydratase (pheA) feedback inhibited by phenylalanine and a chorismate mutase-prephenate dehydrogenase (tyrA) feedback inhibited by tyrosine (9,10); in both cases the Nterminal part of the bifunctional enzyme carries the chorismate mutase activity (19,26).…”

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A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

et al. 1989

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The Saccharomyces cerevisiae ARO7 gene product chorismate mutase, a single-branch-point enzyme in the aromatic amino acid biosynthetic pathway, is activated by tryptophan and subject to feedback inhibition by tyrosine. The ARO7 gene was cloned on a 2.05-kilobase EcoRI fragment. Northern (RNA) analysis revealed a 0.95-kilobase poly(A)+ RNA, and DNA sequencing determined a 771-base-pair open reading frame capable of encoding a protein of 256 amino acids. In addition, three mutant alleles of ARO7 were cloned and sequenced. These encoded chorismate mutases which were unresponsive to tyrosine and tryptophan and were locked in the on state, exhibiting a 10-fold-increased basal enzyme activity. A single base pair exchange resulting in a threonine-to-isoleucine amino acid substitution in the C-terminal part of the chorismate mutase was found in all mutant strains. In contrast to other enzymes in this pathway, no significant homology between the monofunctional yeast chorismate mutase and the corresponding domains of the two bifunctional Escherichia coli enzymes was found.In the yeast Saccharomyces cerevisiae, the biosynthesis of aromatic amino acids is regulated either at the transcriptional or at the enzyme level. At the transcriptional level, the general control system is known to regulate at least 30 structural amino acid genes in various pathways, among them most of the ARO and TRP genes. This transcriptional control responds to amino acid starvation and results in an increased transcription rate of these genes through binding of the activator protein GCN4 (36). In contrast to many bacteria, no aromatic amino acid-specific regulation is known at the transcriptional level.At least four ARO and TRP gene products are also or exclusively regulated at the enzyme level (Fig. 1). The two isoenzymes 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 4.1.2.15) encoded by the genes AR03 and AR04 control the entrance of the shikimate pathway and are subject to feedback inhibition by the pathway end products phenylalanine and tyrosine, respectively (37). The TRP2 gene product anthranilate synthase (EC 4.1.3.27) and the AR07 gene product chorismate mutase (EC 5.4.99.5)

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Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product

Abstract: Fragments of the tyrA gene of Escherichia coli, when suitably engineered, can express either the chorismate mutase activity or the prephenate dehydrogenase activity without the other.

Cited by 24 publications

References 15 publications

Monofunctional Chorismate Mutase from Bacillus subtilis: FTIR Studies and the Mechanism of Action of the Enzyme

Monofunctional Chorismate Mutase from Bacillus subtilis: FTIR Studies and the Mechanism of Action of the Enzyme

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

A single point mutation results in a constitutively activated and feedback-resistant chorismate mutase of Saccharomyces cerevisiae

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