Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli

Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):639-48. doi: 10.1042/bj3310639.


Cystathionine gamma-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the gamma-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine gamma-synthase from Arabidopsis thaliana has been cloned and used to overexpress the enzyme in Escherichia coli. The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5'-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 microM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine gamma-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint gamma-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the gamma-replacement reaction and approx. 1/1500 as fast as the gamma-elimination catalysed by the E. coli enzyme. This huge difference could be attributed to the inability of the A. thaliana cystathionine gamma-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient gamma-elimination reaction catalysed by the enterobacterial enzyme.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / pharmacology
  • Amino Acid Sequence
  • Arabidopsis / enzymology*
  • Binding Sites
  • Carbon-Oxygen Lyases / chemistry
  • Carbon-Oxygen Lyases / genetics*
  • Carbon-Oxygen Lyases / metabolism*
  • Cloning, Molecular
  • DNA, Complementary
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / genetics*
  • Gene Expression*
  • Glycine / analogs & derivatives
  • Glycine / pharmacology
  • Homoserine / analogs & derivatives
  • Homoserine / metabolism
  • Homoserine / pharmacology
  • Kinetics
  • Lysine / metabolism
  • Macromolecular Substances
  • Methionine / biosynthesis
  • Molecular Sequence Data
  • Pyridoxal Phosphate / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • Alkynes
  • DNA, Complementary
  • Enzyme Inhibitors
  • Macromolecular Substances
  • Recombinant Proteins
  • O-phosphohomoserine
  • Pyridoxal Phosphate
  • propargylglycine
  • Homoserine
  • Methionine
  • O-succinylhomoserine (thiol)-lyase
  • Carbon-Oxygen Lyases
  • Lysine
  • Glycine