Methyl‐coenzyme M reductase (MCR) catalyzes the methane‐forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post‐translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME‐1 cluster were also analyzed.

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H4SPT) rather than tetrahydrofolate (H4F) as a pterin C1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H4F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N5,N10-methylene-H4F dehydrogenase/N5,N10-methenyl-H4F cyclohydrolase) and GlyA (serine:H4F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H4F and H4F, respectively (apparent Km below 5 microM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H4F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H4F. From the p-aminobenzoic acid requirement, an intracellular H4F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H4SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H4SPT and H4F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C1 pools in these methanogens.

The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of C␤ of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C 1 ) units for several essential biosynthetic processes. In many Archaea, C 1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability.Serine hydroxymethyltransferase (SHMT, 1 EC 2.1.2.1) catalyzes the reversible transfer of C␤ of serine to tetrahydropteroylglutamate (H 4 PteGlu) to form glycine and 5,10-methylene-H 4 PteGlu. In Eukarya and Eubacteria, H 4 PteGlu functions as a carrier of C 1 units in several oxidation states, which are used in the biosynthesis of important cellular components, such as purines and thymidylate, in the regeneration of methionine from homocysteine or, in acetogenic bacteria, in the synthesis of acetyl-CoA. The reaction catalyzed by SHMT represents in these organisms one of the major loading routes of C 1 units onto the folate carrier (1). In methanogens and several other Archaea, C 1 fragments from formyl to methyl oxidation levels are carried by tetrahydromethanopterin (H 4 MPT), a pterin-containing compound involved in methanogenesis. Although H 4 PteGlu and H 4 MPT are structurally similar in their pterin-like portion (Fig. 1) and in the role as C 1 units carriers, they are functionally distinct. H 4 MPT does not appear to be suited to most of the biosynthetic functions of H 4 PteGlu. Moreover, the biosynthetic pathways of the two carriers have few, if any, homologies, suggesting the possibility of separate evolutionary origins (2). In the metabolism of folates, SHMT represents a...

The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-L-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing ␦-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.