Two putative malate dehydrogenase genes, MJ1425 and MJ0490, from Methanococcus jannaschii and one from Methanothermus fervidus were cloned and overexpressed in Escherichia coli, and their gene products were tested for the ability to catalyze pyridine nucleotide-dependent oxidation and reduction reactions of the following ␣-hydroxy-␣-keto acid pairs: (S)-sulfolactic acid and sulfopyruvic acid; (S)-␣-hydroxyglutaric acid and ␣-ketoglutaric acid; (S)-lactic acid and pyruvic acid; and 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid and 1-oxo-1,3,4,6-hexanetetracarboxylic acid. Each of these reactions is involved in the formation of coenzyme M, methanopterin, coenzyme F 420 , and methanofuran, respectively. Both the MJ1425-encoded enzyme 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 nucleotides. Both the M. fervidus and the MJ1425-encoded enzyme catalyzed the NAD ؉ -dependent oxidation of (S)-sulfolactate to sulfopyruvate. The MJ1425-encoded enzyme also catalyzed the NADH-dependent reduction of ␣-ketoglutaric acid to (S)-hydroxyglutaric acid, a component of methanopterin. Neither of the enzymes reduced pyruvate to (S)-lactate, a component of coenzyme F 420 . Only the MJ1425-encoded enzyme was found to reduce 1-oxo-1,3,4,6-hexanetetracarboxylic acid, and this reduction occurred only to a small extent and produced an isomer of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid that 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.The biosynthesis of the methanogenic cofactors coenzyme M (2-mercaptoethanesulfonic acid), methanopterin, coenzyme F 420 , and methanofuran c (Fig. 1) requires the generation of an ␣-hydroxy acid that either becomes a component in the final structure or serves as an intermediate in the formation of the coenzyme. In the case of coenzyme M, (S)-sulfolactate, formed from phosphoenolpyruvate (PEP) and bisulfite, is an intermediate in the biosynthesis (28-30). In the case of methanopterin (24, 25) and several related modified folates (33,34,36), (S)-hydroxyglutaric acid (23) is incorporated into the coenzyme during its biosynthesis (32, 35). For coenzyme F 420 (6) and its ␥-polyglutamate derivatives (7, 8, 18), (S)-hydroxypropionic acid (S-lactic acid) becomes a part of the final structure. Finally, two (1R)-diastereomers of 1-hydroxy-1,3,4,6-hexanetetracarboxylic acid (HHTCA) serve as intermediates in the biosynthesis of the 1,3,4,6-hexanetetracarboxylic acid (HTCA) moiety of methanofuran (17; unpublished results), and another diastereomer of HHTCA [(1S)-HHTCA] is a component of metha...

The protein product of the Methanococcus jannaschii MJ0768 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to catalyze the GTP-dependent addition of two l-glutamates to the l-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) to form F(420)-0-glutamyl-glutamate (F(420)-2). Since the reaction is the fifth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofE, the product of the cofE gene. Gel filtration chromatography indicates CofE is a dimer. The enzyme has no recognized sequence similarity to any previously characterized proteins. The enzyme has an absolute requirement for a divalent metal ion and a monovalent cation. Among the metal ions tested, a mixture of Mn(2+), Mg(2+), and K(+) is the most effective. CofE catalyzes amide bond formation with the cleavage of GTP to GDP and inorganic phosphate, likely involving the activation of the free carboxylate group of F(420)-0 to give an acyl phosphate intermediate. Evidence for the occurrence of this intermediate is presented. A reaction mechanism for the enzyme is proposed and compared with other members of the ADP-forming amide bond ligase family.

The protein product of the Methanococcus jannaschii MJ1256 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to be involved in coenzyme F(420) biosynthesis. The protein catalyzes the transfer of the 2-phospholactate moiety from lactyl (2) diphospho-(5')guanosine (LPPG) to 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) with the formation of the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) and GMP. On the basis of the reaction catalyzed, the enzyme is named LPPG:Fo 2-phospho-L-lactate transferase. Since the reaction is the fourth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofD, the product of the cofD gene. The transferase requires Mg(2+) for activity, and the catalysis does not appear to proceed via a covalent intermediate. To a lesser extent CofD also catalyzes a number of additional reactions that include the formation of Fo-P, when the enzyme is incubated with Fo and GDP, GTP, pyrophosphate, or tripolyphosphate, and the hydrolysis of F(420)-0 to Fo. All of these side reactions can be rationalized as occurring by a common mechanism. CofD has no recognized sequence similarity to any previously characterized enzyme.

The biochemical route for the formation of the phosphodiester bond in coenzyme F(420), one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F(420) structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactate represents a new natural product that was chemically identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc. jannaschii. Incubation of cell extracts of both M. thermophila and Mc. jannaschii with [hydroxy-(18)O, carboxyl-(18)O(2)]lactate and GTP produced 2-phospho-L-lactate with the same (18)O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex G-25 purified cell extracts of M. thermophila or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F(420)-0 (F(420) with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F(420)-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extracts with L-[U-(14)C]lactate, [U-(14)C]2-phospho-L-lactate, or [8-(3)H]GTP were not successful owing to the instability of these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 min at 50 degrees C (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesis could be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F(420)-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F(420) follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B(12) and phospholipids.

The products of two adjacent genes in the chromosome of Methanococcus jannaschii are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in Streptomyces wedmorensis. These two M. jannaschii genes were recombinantly expressed in Escherichia coli, and their gene products were tested for the ability to catalyze the decarboxylation of a series of ␣-ketoacids. Both subunits are required to form an ␣ 6 ␤ 6 dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The M. jannaschii sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.Coenzyme M (2-mercaptoethanesulfonic acid) was originally characterized as one of several coenzymes involved in the formation of methane in the methanoarchaea (14). Recently it has been shown to function as a coenzyme in the bacterial metabolism of aliphatic alkenes (1). Despite the fact that the pathway for its biosynthesis (Fig. 1) has been known for a number of years (35-37), no genes or enzymes involved in its biosynthesis have been identified. One of the steps in the biosynthesis of coenzyme M, the decarboxylation of sulfopyruvate to sulfoacetaldehyde, is chemically very analogous to the decarboxylation of phosphonopyruvate that occurs in the biosynthesis of natural products containing a C-P bond. Among these are fosfomycin (19), phosphinothricin (30), and bialaphos (24), each produced by various species of Streptomyces. The sequence homology among the Streptomyces genes for phosphonopyruvate decarboxylase (30), the genes contained in Methanococcus jannaschii MJ0256 (7), and the Methanobacterium thermoautotrophicum genes MT1206 and MT1207 (31) (Fig. 2) has prompted us to clone and overexpress the two proteins encoded by the MJ0256 open reading frame. This work has established that these two protein products catalyze the decarboxylation of sulfopyruvate to form sulfoacetaldehyde and CO 2 . The enzyme has been named sulfopyruvate decarboxylase, and the gene has been named comDE, to indicate that the enzymatic reaction is the fourth expected in the biosynthesis of coenzyme M from phosphoenol pyruvate and bisulfite. From sequence comparisons with other similar thiamine-PP (TPP)-dependent enzymes (Fig. 2) and from the nature of the reaction catalyzed, it is clear that this enzyme is a TPP-dependent enzyme since it contains the TPP-binding motif (DGDGSILMNLGSLSTIGYMNPKNYILVIIDN) (18). Unexpectedly, the enzyme was found to be readily inactivated by exposure to oxygen. MATERIALS AND METHODSChemicals. Sulfopyruvate and sulfoacetaldehyde were prepared as previously described (36, 37). Zhibing Lu, Department of Chemistry, University of New Mex...