Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum

Biol Chem. 1999 Oct;380(10):1237-42. doi: 10.1515/BC.1999.157.

Abstract

Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carbon-Oxygen Lyases / chemistry*
  • Carbon-Oxygen Lyases / isolation & purification
  • Carbon-Oxygen Lyases / metabolism*
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Kinetics
  • Macromolecular Substances
  • Molecular Sequence Data
  • Plants, Toxic*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Tobacco / enzymology*
  • Tobacco / genetics

Substances

  • Macromolecular Substances
  • Recombinant Proteins
  • O-succinylhomoserine (thiol)-lyase
  • Carbon-Oxygen Lyases