A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature‐regulated way

Abstract

In Saccharomyces cerevisiae, aspartate kinase (the HOM3 product) regulates the metabolic flux through the threonine biosynthetic pathway through feedback inhibition by the end product. In order to obtain a strain able to produce threonine in a controlled way, we have isolated a mutant allele (HOM3-ts31d ) that gives rise to a deregulated aspartate kinase. This allele has been isolated as an extragenic suppressor of ilv1, which confers an Ilv + phenotype at 37 • C but not at 22 • C. We have stated that at high temperature the mutant aspartate kinase is slightly more deregulated and shows a higher specific activity, inducing threonine accumulation. The HOM3-ts31d allele carries a mutation that leads to a Ser399 → Phe substitution in the postulated regulatory region of the enzyme. We have detected other changes in the nucleotide sequence but they are also present in the parental strain, reflecting the genetic differences between different wild-type strains. A sequence comparison among all the reported mutant aspartate kinases suggests that not all residues involved in regulation of the activity are clustered in the so-called regulatory domain, as is the case of that mutated in AK-R7, another deregulated aspartate kinase obtained with the same strategy of ilv1 suppression.

Horizontally acquired DAP pathway as a unit of self-regulation

“…These two enzymes are most closely related to homologues from Saccharomyces cerevisiae and Escherichia coli, both evolutionarily and structurally, among all organisms with known regulatory site information. The enzymes from S. cerevisiae and E. coli are under different regulations; the former is allosterically regulated by threonine and contains four binding sites for allosteric effectors (Martin-Rendon et al, 1993;Arevalo-Rodriguez et al, 1999;Velasco et al, 2005), whereas the latter contains seven binding sites and is regulated by lysine (Kikuchi et al, 1999;Ogawa-Miyata et al, 2001). Our motif-based analyses show that the same or similar amino acids are present in the regulatory sites of the two Monosiga aspartate kinases (Fig.…”

“…The structure of aspartate kinase is characterized by a conserved N‐terminal amino acid kinase domain and one or more C‐terminal ACT regulatory domains (Velasco et al. , 2005).…”

“…, 1993; Arevalo‐Rodriguez et al. , 1999; Velasco et al. , 2005), whereas the latter contains seven binding sites and is regulated by lysine (Kikuchi et al.…”

“…The structure of aspartate kinase is characterized by a conserved N-terminal amino acid kinase domain and one or more C-terminal ACT regulatory domains (Velasco et al, 2005). In Monosiga, the regular aspartate kinase contains two ACT domains, but only a single domain is present in the bifunctional enzyme.…”

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

“…8A. It appears that in this case additional residues important for threonine allostery are located remote from the ACT domains in the catalytic domain (87). Ectoine-associated Ask species are all present in CG-02, and they also exhibit remnants of residues deemed important for This group is known to be subject to synergistic inhibition by lysine and threonine (46), so derivation from a lysine-sensitive Ask seems reasonable.…”