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.
Institut für Pharmazeutische Biologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Federal Republic Germany.
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Igbavboa
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Leistner
D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology; D006836:Hydro-Lyases / Q000378:metabolism*; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009168:Mycobacterium phlei / Q000201:enzymology; D011549:Pseudomonas / Q000201:enzymology; D011865:Radioisotope Dilution Technique; D014316:Tritium; D014812:Vitamin K / Q000096:biosynthesis*
D002250:Carbon Radioisotopes; D014316:Tritium; D014812:Vitamin K; D007521:Isocitrate Dehydrogenase; D006836:Hydro-Lyases; C021853:naphthoate synthetase