O-Succinylhomoserine as an Intermediate in the Synthesis of Cystathionine by Escherichia coli

Abstract

Further evidence that cystathionine is a precursor of methionine synthesis in E. coli was obtained by showing its synthesis by cell-free extracts of an auxotroph which required homocysteine or methionine for growth. Succinate, as well as homo-serine and cysteine, was an obligatory substrate and maximal synthesis was dependent on the further addition of glucose, adenosine-triphosphate and coenzyme-A. The reaction was further studied by using mixtures of extracts of auxotrophs of which each alone was unable to synthesize cystathionine. At least two enzymes were required the first, present in one strain, formed a heat-stable intermediate from homoserine and succinate; while the second, present in the other strain, converted this intermediate to cystathionine in the presence of cysteine. The intermediate was formed enzymically from homoserine and succinyl-coenzyme-A alone and was ninhydrin-positive; this suggested that it might be O-succinylhomoserine. This compound was prepared by reaction of succinyl-chloride or succinic-anhydride with homoserine in the presence of perchloric-acid; its chromatographic and chemical properties were closely similar to those of the intermediate. The synthetic product, like that formed enzymically, gave cystathionine when incubated with cysteine and a cell-free extract of the relevant E. coli auxotroph. It is concluded that O-succinylhomoserine is an intermediate in the formation of cystathionine, and consequently of methionine by E. coli.