Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis

The enzymic conversion of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid (i.e. o-succinylbenzoic acid) to 1,4-dihydroxy-2-naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two-step process, was investigated using chirally labelled samples of the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate-lyase (EC 4.1.3.1), 2-oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4-(2'-carboxyphenyl)-4-oxobutyrate synthase system and 4-(2'-carboxyphenyl)-4-oxobutyrate: CoA ligase. Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiomeric samples of 2-oxoglutarate enantiotopically labelled at C-3. These samples were converted enzymically to succinate with retention of configuration at C-2 and C-3, and to 4-(2'-carboxyphenyl)-4-oxobutyric acid with retention of configuration at C-3. Isocitrate glyoxylate-lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2-oxoglutarate enantiotopically tritiated at C-4 or at C-3 and C-4. The four variously labelled samples of 2-oxoglutarate were enzymically converted to the coenzyme A ester of 4-(2'-carboxyphenyl)-4-oxobutyric acid. The resulting variously labelled coenzyme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRe atom of the oxobutyric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C-3 of 1,4-dihydroxy-2-naphthoic acid. The second step in this ring closure reaction is the rate-limiting step.