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

Abstract

SUMMARYFurther evidence that cystathionine is a precursor of methionine synthesis in Escherichia coli was obtained by showing its synthesis by cellfree extracts of an auxotroph which required homocysteine or methionine for growth. Succinate, as well as homoserine 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.

Methionine genes and enzymes of Salmonella typhimurium

“…Results obtained support the existence of the 5 methionine genes originally identified (metA, B, C, E and F) and have revealed another (metG). Parallel work on the enzyme deficiencies of similar Escherichia coli mutants and representative S. tphimurium mutants (Rowbury, 1964a, b; Rowbury and Woods, 1964;Wijesundera and Woods, 1962;Woods, Foster and Guest, 1965;Foster, personal communication) has permitted the firm establishment of the same pathway of methionine synthesis in these two organisms ( fig. ).…”

Biosynthesis of Protein Amino Acids in Plant Tissue Culture IV Isotope Competition Experiments using Glucose-U-14C and Potential Intermediates

“…Cystathioniine and( homocvsteiine als,o contribuited carbon to methionine thuis otlliniing the pathway of methionine biosynthesis. Rowbury and \Vood (26) and Delavier-Kltitchko and Flavin (4) Ouir resu-lts show that dturing growth of the Rose tisstue, a-keto-isovaleric acid is ain intermediate in the biosynthesis of valine and leuiciine. This complements the demonstration of the enzymes for the synthesis oaf letucine from a-keto isovaleric acid in maize embryos (25).…”

The Regulation of Cystathionine Formation in Escherichia coli

“…of 14C2,,-succinate added. Activity was assessed by the incorporation of I4C into 0-succinylhomoserine (separated from succinate on columns of Dowex-1 resin as described by Rowbury & Woods, 1964b). The synthesis of O-succinylhomoserine by whole organisms of E. coli strain 7/9 (incubated in medium GL) was also assayed by uptake of 14C-succinate.…”

“…The overall conversion of homoserine + cysteine to cystathionine by extracts from Escherichia coli strain 26/18 was assayed as described by Rowbury & Woods (1964b). Incubation was for 3-4 hr at 37' in solution A which contained (in 3 ml.…”