Identification of an archaeal 2-hydroxy acid dehydrogenase catalyzing reactions involved in coenzyme biosynthesis in methanoarchaea

Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococ cus jannaschii and one from Methanothermus fervidus were cloned and overexp ressed in Escherichia coli, and their gene products were tested for the abi lity to catalyze pyridine nucleotide-dependent oxidation and reduction reac tions of the following alpha-hydroxy-alpha-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-alpha-hydroxyglutaric acid and cx-ketoglut aric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanete tracarboxylic acid and 1-oxo-1,3,4,6-hexanetetracarboxylic acid. Each of th ese reactions is involved in the formation of coenzyme M, methanopterin, co enzyme F-420, and methanofuran, respectively. Both the MJ1425-encoded enzym e and the MJ0490-encoded enzyme were found to function to different degrees as malate dehydrogenases, reducing oxalacetate to (S)-malate using either NADH or NADPH as a reductant, Both enzymes were found to use either NADH or NADPH to reduce sulfopyruvate to (S)-sulfolactate, but the V-max/K-m value for the reduction of sulfopyruvate by NADH using the MJ1425-encoded enzyme was 20 times greater than any other combination of enzymes and pyridine nu cleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD(+)-dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ142 5-encoded enzyme also catalyzed the NADH-dependent reduction of alpha-ketog lutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Nei ther of the enzymes reduced pyruvate to (S)-lactate, a component of coenzym e F-420. Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-h exanetetracarboxylic acid, and this reduction occurred only to a small exte nt and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid t hat is not involved in the biosynthesis of methanofuran c. We conclude that the MJ1425-encoded enzyme is likely to be involved in the biosynthesis of both coenzyme M and methanopterin.