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

Abstract

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)

Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum

“…EcCM exists as a fusion protein in E. coli and is subject to allosteric inhibition only by pathwayspecific inhibitors (Prakash et al, 2005). The ScCM of S. cerevisiae and AnCM of Aspergillus nidulans are inhibited by Tyr and Phe and are stimulated by Trp (Krappmann et al, 1999;Schmidheini et al, 1989). Similar to MtbCM (Kim et al, 2006), CM0819 in C. glutamicum is not subject to any regulation by aromatic amino acids.…”

Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase

“…A constitutively activated yeast CM is known in which the last residue of this particular loop (Thr-226) is substituted by an isoleucine residue. This residue locks the enzyme structure in the allosteric R state, which completely prevents inhibition or further activation (6). The end of this loop, however, is not as strongly conserved among these homologous CMs.…”

Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

“…It is of interest to note that mutagenesis studies demonstrated that threonine residue 278 was essential for prephenate dehydratase activity (Baldwin & Davidson, 1981). While it appears that the prephenate dehydratases may share a common ancestor, a monophyletic origin of chorismate mutase enzymes is not obvious since the CM-Fs of B. subtilis (Gray et al, 1990) and Saccharomyces cerevisiae (Schmidheini et al, 1989) showed marginal to no similarity to the P. stutzeri P-protein, the E. coli P-protein, the E. coli Tprotein, or to each other. The sequence of the gene encoding the bifunctional DAHP synthase : chorismate mutase from B. subtilis 168 (Hoch & Nester, 1973;Nasser & Nester, 1967) may provide clues about the evolution of chorismate mutase.…”