Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase ofSaccharomyces cerevisiae

Abstract

The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine‐induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation. One of the mutants has an aspartate kinase which is 30‐fold more strongly inhibited by threonine than the wild‐type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine‐phenylalanine‐glycine‐glycine (KFGG) stretch present in the N‐terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved in substrate binding. Mutagenesis of a HOM3 region centred in the KFGG‐coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback‐resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme. Copyright © 1999 John Wiley & Sons, Ltd.

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

“…In a second step, 5-FOA resistant colonies were selected; among the Ura − transformants were some Hom − colonies. Deletion at the HOM3 locus was verified by diagnostic PCR with the primers HOMpriA and HOMpriB (Arévalo-Rodríguez et al, 1999).…”

“…Plasmid pMAC33 h was obtained from pMAC32wt by removing the whole HOM3 coding region (Arévalo-Rodríguez et al, 1999). The wild-type HOM3-87wt and the mutant HOM3-R7 alleles were cloned by the gap-repair method (Rothstein, 1985) by transforming strains SG193 and SG212, respectively, with plasmid pMAC33 h, previously linearized by digestion with HindIII, and selecting Ura + transformants.…”

“…The whole ORF of HOM3-R7 (bp 365-1948 of the published HOM3 sequence; Rafalski and Falco, 1988) was sequenced using primers OLIGO-I, -II, -III, -IV, -V, -VI and -VII (Arévalo-Rodríguez et al, 1999). The ORF of the HOM3-87wt allele was partially sequenced using primers MGI and OLIGOIV (Arévalo-Rodríguez et al, 1999).…”

“…The ACT domain has been found in enzymes that are regulated by amino acids or purines and is involved in regulation by these effectors (Chipman and Shaanan, 2001). In Figure 5, only aspartate kinases for which single mutations affecting enzymatic activity or regulation have been described (Arévalo-Rodríguez et al, 1999;Bareich and Wright, 2003;Farfán, 1997;Follettie et al, 1993;Hernando-Rico et al, 2001;Kalinowski et al, 1991;Kikuchi et al, 1999;Marco-Marin et al, 2003;Martín-Rendón et al, 1993;Ogawa-Miyata et al, 2001;Ohnishi et al, 2002;Omori et al, 1993;Paris et al, 2003) are shown. Most but not all of those mutations affecting regulation are located in the ACT domains.…”

“…A few of them are in the amino acid kinase domain, viz. E282D and E279A in S. cerevisiae (this work; Bareich and Wright, 2003) (12) I522A (12) Q524 (12) AtAK-HD S301Y (8) G345D (8) AlAK E164K (6) K8R (5) E119D (5) R198K (5) D202E (5) E250K (7) G323D (7,6) M318I (6) L325F (6) T352I (7,6) V347M (6) T344M (7) S345L (7,6) EcAKIII S301Y (9) T311Y ( Bareich and Wright, 2003;(2) Arévalo-Rodríguez et al, 1999;(3) Martín-Rendón et al, 1993;(4) Farfán, 1997;(5) Marco-Marin et al, 2003;(6) Ogawa-Miyata et al, 2001;(7) Kikuchi et al, 1999;(8) Hernando-Rico et al, 2001;(9) Kalinowski et al, 1991;(10) Ohnishi et al, 2002;(11) Follettie et al, 1993;(12) Paris et al, 2003;(13) Omori et al, 1993Omori et al, et al, 1999Marco-Marin et al, 2003;O...…”