Methionine biosynthesis in higher plants: biochemical and molecular characterization of the transsulfuration pathway enzymes

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In plants, the transfer of the sulfur atom between cysteine and homocysteine, the direct precursor of methionine, is ensured by two chloroplastic enzymes, cystathionine γ-synthase and cystathionine β-lyase. These proteins have been purified to homogeneity from spinach chloroplasts and their biochemical properties determined. Cystathionine γ-synthase and cystathionine β-lyase are tetramers and are typical pyridoxal 5′-phosphate-dependent proteins. These enzymes are targets for the potent inhibitors of methionine synthesis that are lethal for plants. An Arabidopsis thaliana cDNA encoding chloroplastic cystathionine β-lyase was isolated by functional complementation of a bacterial mutant and cloned in a pET expression vector in order to transform Escherichia coli cells. Preliminary observations of the active site of the purified recombinant enzyme have been performed by characterization of the interaction between i) pyridoxal 5′-phosphate and the polypeptide chain, and ii) the active site-directed inhibitor aminoethoxyvinylglycine and the bound cofactor. This study will be developed further by crystallographic analyses.