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Escherichia coli K12 and a mutant of E. coli (viz., AN 154) as well as Aerobacter aerogenes 62-1 (i.e., Klebsiella pneumoniae) were used as sources of the enzyme catalyzing the formation of o-succinylbenzoic acid (OSB) from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate. The product of the reaction (OSB) was identified by HPLC before and after derivatization to the methylester, dilactone, and coenzyme A ester. OSB synthase and alpha-ketoglutarate dehydrogenase are similar in that both decarboxylate alpha-ketoglutarate in the presence of thiamine pyrophosphate but the enzyme systems can be separated easily by several methods. Reexamination of mutants E. coli AN 154 and AN 191 showed that these mutants are leaky, rather than blocked, between chorismic acid and isochorismic acid. This finding, together with the observation that isochorismic acid rather than chorismic acid is the substrate of OSB synthase, invalidates previous assumptions on the reaction initiating vitamin K2 biosynthesis.

Cell-free extracts of various strains of Escherichia 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-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 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 a 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 one gene product and converted to OSB by the second gene product.

A men- mutant of Escherichia coli, AN 209, which accumulates o-succinylbenzoic acid, has been used for a direct study of the biosynthesis of this benzenoid compound. Samples of labeled glutamic acids were added to growth media, and the o-succinylbenzoic acid was isolated and converted to a dimethyl derivative. This dimethyl derivative was purified on thin-layer chromatograms and by gas chromatography. When the glutamic acid used as precursor contained 14C at position 5, or was uniformly labeled, the dimethyl o-succinylbenzoate contained radioactivity (as shown by radiogas chromatography). However, from [1-14C]glutamate, the dimethyl o-succinylbenzoate was without radioactivity. Hence, in the biosynthesis of o-succinylbenzoate, carbon atom 1 of glutamate is lost, and carbon atoms 2-5 are retained. It was also shown that this mutant lacked the enzyme dihydroxynaphthoic acid synthase. It should, therefore, continue to be classified as a menB mutant, rather than as a member of the newly created menE group (lacking o-succinylbenzoate-CoA synthetase).

Cell-free preparations have been obtained from Escherichia coli AN 154 which catalyze the conversion of chorismic acid and alpha-ketoglutaric acid to o-succinylbenzoic acid. This result constitutes the first experimental verification of the committed step in menaquinone biosynthesis. The enzymatic biosynthesis of o-succinylbenzoic acid was demonstrated in experiments utilizing [U-14C]alpha-ketoglutaric acid as substrate. Following purification of O-succinylbenzoic acid and conversion to the dimethyl derivative, the presence of 14C was shown by scanning of thin layer chromatograms, and by radiogas chromatography. Proof for the formation of o-succinylbenzoic acid was also obtained by combined gas chromatography/mass spectrometry. The formation of o-succinylbenzoic acid in these extracts required the presence of thiamin pyrophosphate; when this cofactor was omitted from the incubations, there was a substantial diminution in the incorporation of radioactivity from alpha-ketoglutarate into o-succinylbenzoic acid (from 4- to 8-fold). These results support the suggestion (Campbell, I. M. (1969) Tetrahedron Lett. 4777-4780) that the three-carbon unit of lawsone and, hence by inference, the four-carbon side chain of o-succinylbenzoic acid, is derived from the thiamin pyrophosphate adduct of succinic semialdehyde (likely in the anion form). The activated form of succinic semialdehyde is derived by the action of the first enzyme of the alpha-ketoglutarate dehydrogenase complex or by a similar decarboxylase enzyme.

The enzyme system (OSB2 synthase) catalyzing the synthesis of o-succinylbenzoic acid from isochorismic acid and alpha-ketoglutaric acid in the presence of thiamine pyrophosphate was isolated from Escherichia coli AN 154 and characterized. The purification factor of the enzyme did not increase during column chromatography on Sephadex G-200 or chromatofocusing, suggesting that the OSB synthase is labile. Chromatography on DEAE-Sephadex A-50 or A-25 showed that an enzyme activity separated from fractions containing OSB synthase that decarboxylates alpha-ketoglutarate. This activity is provisionally referred to as the decarboxylating "subunit" or decarboxylating activity of OSB synthase. Both the "subunit" and the holoenzyme were characterized with respect to pH optimum, temperature optimum, and KM values. The OSB synthase loses all activity during treatment with EDTA and activity is most efficiently restored with Mn2+. The activity of the decarboxylating subunit did not depend on Mn2+. When the decarboxylating fraction was incubated with alpha-ketoglutarate and thiamine pyrophosphate, succinic semialdehyde could be isolated as its hydrazone. After treatment of the incubation mixture with phosphorylase a compound was isolated which is most likely the thiamine adduct of succinic semialdehyde.