Publications

• Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene.

  Lee M., Leustek T.

  Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana tha ... >> More

  Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/-0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro. << Less

  Arch. Biochem. Biophys. 372:135-142(1999) [PubMed] [EuropePMC]

• Convergent evolution of coenzyme M biosynthesis in the Methanosarcinales: cysteate synthase evolved from an ancestral threonine synthase.

  Graham D.E., Taylor S.M., Wolf R.Z., Namboori S.C.

  The euryarchaeon Methanosarcina acetivorans has no homologues of the first three enzymes that produce the essential methanogenic coenzyme M (2-mercaptoethanesulfonate) in Methanocaldococcus jannaschii. A single M. acetivorans gene was heterologously expressed to produce a functional sulfopyruvate ... >> More

  The euryarchaeon Methanosarcina acetivorans has no homologues of the first three enzymes that produce the essential methanogenic coenzyme M (2-mercaptoethanesulfonate) in Methanocaldococcus jannaschii. A single M. acetivorans gene was heterologously expressed to produce a functional sulfopyruvate decarboxylase protein, the fourth canonical enzyme in this biosynthetic pathway. An adjacent gene, at locus MA3297, encodes one of the organism's two threonine synthase homologues. When both paralogues from this organism were expressed in an Escherichia coli threonine synthase mutant, the MA1610 gene complemented the thrC mutation, whereas the MA3297 gene did not. Both PLP (pyridoxal 5'-phosphate)-dependent proteins were heterologously expressed and purified, but only the MA1610 protein catalysed the canonical threonine synthase reaction. The MA3297 protein specifically catalysed a new beta-replacement reaction that converted L-phosphoserine and sulfite into L-cysteate and inorganic phosphate. This oxygen-independent mode of sulfonate biosynthesis exploits the facile nucleophilic addition of sulfite to an alpha,beta-unsaturated intermediate (PLP-bound dehydroalanine). An amino acid sequence comparison indicates that cysteate synthase evolved from an ancestral threonine synthase through gene duplication, and the remodelling of active site loop regions by amino acid insertion and substitutions. The cysteate product can be converted into sulfopyruvate by an aspartate aminotransferase enzyme, establishing a new convergent pathway for coenzyme M biosynthesis that appears to function in members of the orders Methanosarcinales and Methanomicrobiales. These differences in coenzyme M biosynthesis afford the opportunity to develop methanogen inhibitors that discriminate between the classes of methanogenic archaea. << Less

  Biochem. J. 424:467-478(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and properties of threonine synthetase of Neurospora.

  FLAVIN M., SLAUGHTER C.

  J Biol Chem 235:1103-1108(1960) [PubMed] [EuropePMC]

• Substrate specificity and identification of functional groups of homoserine kinase from Escherichia coli.

  Huo X., Viola R.E.

  Homoserine kinase, an enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the conversion of L-homoserine to L-homoserine phosphate. This enzyme has been found to have broad substrate specificity, including the phosphorylation of L-homoserine analogs where the ... >> More

  Homoserine kinase, an enzyme in the aspartate pathway of amino acid biosynthesis in Escherichia coli, catalyzes the conversion of L-homoserine to L-homoserine phosphate. This enzyme has been found to have broad substrate specificity, including the phosphorylation of L-homoserine analogs where the carboxyl functional group at the alpha-position has been replaced by an ester or by a hydroxymethyl group. Previous pH profile studies [Huo. X., & Viola, R. E. (1996) Arch. Biochem. Biophys. 330, 373-379] and chemical modification studies have suggested the involvement of histidinyl, lysyl, and argininyl residues in the catalytic activity of the enzyme. With the assistance of sequence alignments, several potential amino acids have been targeted for examination. Site-directed mutagenesis studies have confirmed a role for arginine-234 in the binding of the carboxyl group of L-homoserine, and the involvement of two histidine at the homoserine binding site. Mutations at these sites have led to the decoupling of the kinase activity from an inherent ATPase activity in the enzyme, and suggest the presence of independent domains for the binding of each substrate in homoserine kinase. << Less

  Biochemistry 35:16180-16185(1996) [PubMed] [EuropePMC]