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

Minsang Lee, Thomas Leustek

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

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 H 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 μmol min-1 mg-1 protein at pH 8.5 and 37°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 Mg2+ 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.

Original languageEnglish (US)
Pages (from-to)135-142
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume372
Issue number1
DOIs
StatePublished - Dec 1 1999

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Keywords

  • Arabidopsis thaliana
  • Cystathionine-γ-synthase
  • Homoserine kinase
  • O- succinyl-L-homoserine
  • O-acetyl-L-homoserine
  • O-phospho-L-homoserine
  • S-adenosyl-L- methionine
  • Threonine synthase

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