Thiamine pyrophosphate requirement for o-succinylbenzoic acid synthesis in Escherichia coli and evidence for an intermediate

Abstract

Cell-free extracts of various strains of E. coli synthesize the menaquinone biosynthetic intermediate o-succinylbenzoic acid (OSB) when supplied with chorismic acid, 2-ketoglutaric acid and thiamine pyrophosphate (TPP). To assay for OSB synthesis, 2-[U[uniformly labeled]-14C]ketoglutaric acid was used as substrate, and the synthesized OSB was examined by radiogas chromatography (as the dimethyl ester). [U-14C]Shikimic acid also gave rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to radioactive OSB if the cofactors necessary for enzymatic conversion to chorismic acid were added. Use of 2-[1-14C]ketoglutaric acid does not give rise to labeled OSB. In the absence of TPP during the incubations, OSB synthesis was much reduced; these observations are consistent with the proposed role for the succinic semialdehyde-TPP anion as the reagent adding to chorismic acid. Extracts of cells from menC and menD mutants did not form OSB separately but did so in combination. There was evidence for formation of product, X, by extracts of a menC mutant incubated with chorismic acid, TPP and 2-ketoglutaric acid; X was converted to OSB by extracts of a menD mutant. It appears that the intermediate, X, is formed by 1 gene product and converted to OSB by the 2nd gene product.