Cystathionine gamma-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli.
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.
Ravanel
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Laboratoire Mixte CNRS/Rhône-Poulenc (UMR41), Rhône-Poulenc Agrochimie, 14-20 rue Pierre Baizet, 69263 Lyon cedex 09, France.
Gakière
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Job
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Douce
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D000480:Alkynes / Q000494:pharmacology; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005998:Glycine / Q000031:analogs & derivatives / Q000494:pharmacology; D006714:Homoserine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D046911:Macromolecular Substances; D008715:Methionine / Q000096:biosynthesis; D008969:Molecular Sequence Data; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D000480:Alkynes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C011066:O-phosphohomoserine; D011732:Pyridoxal Phosphate; C009055:propargylglycine; D006714:Homoserine; D008715:Methionine; C022696:O-succinylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases; D008239:Lysine; D005998:Glycine