Methanol:5-hydroxybenzinmidazolylcobamide methyltransferase from Methanosarcina barkeri has been purified to approximately 90% homogeneity by ion-exchange chromatography on DEAE-cellulose and QAE-A50 Sephadex columns. The molecular weight, estimated by gel electrophoresis, was found to be 122,000, and the enzyme contained two different subunits with molecular weights of 34,000 and 53,000, which indicates an a213 structure. The enzyme contains three or four molecules of 5-hydroxybenzimidazolylcobamide, which could be removed by treatment of the enzyme with 2-mercaptoethanol or sodium dodecyl sulfate. In both cases the enzyme dissociated into its subunits. For stability, the enzyme required the presence of divalent cations such as Mge+, Mn2+, Sr2+, Ca2+, or Ba2+. ATP, GTP, or CTP was needed in a reductive activation process of the enzyme. This activation was brought about by a mixture of H2, ferredoxin, and hydrogenase, but also by CO, which is thought to reduce the corrinoid chemically. The CO dehydrogenase-like activity of the methyltransferase is discussed. Methanosarcina barkeri is a methanogenic bacterium that can grow on various one-carbon compounds such as C02, methylamines, methanol, and CO and on acetate (3, 10, 11, 23). Growth on methanol has been reported in both the presence and absence of H2; in the latter case the reduction equivalents needed in methanogenesis were derived from the oxidation of part of the methanol to CO2 (11). The reduction of methanol to CH4 in cell-free extracts of M. barkeri was found to depend on the presence of coenzyme M (2-mercaptoethanesulfonic acid; HS-CoM) and ATP under an atmosphere of H2 (12). First, HS-CoM is methylated to 2-(methylthio)ethanesulfonic acid (methylcoenzyme M; CH3S-CoM) (18, 21). CH3S-CoM is subsequently reduced to methane by a methylreductase system that contains an enzyme-bound coenzyme MF430 (7, 8, 13). The involvement of two distinct methyltransferases in the formation of CH3S-CoM from methanol was recently reported (21). Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MT1) binds the methyl group of methanol to a corrinoid bound to this enzyme (22). The enzyme is subject to activation and inactivation. Inactivation is brought about by 02 and other oxidizing agents, and activation is achieved in the presence of ATP and H2 (21). Activation of the partially purified MT, requires also the presence of hydrogenase and ferredoxin and leads to the formation of a Co(I) corrinoid (B12) (22b). The role of the catalytic amount of ATP in this activation has not been elucidated. The second methyltransferase, methylcobalamin:HS-CoM methyltransferase (MT2), is oxygen stable, and ATP is not required in its activity (20). It transfers the methyl group of the bound corrinoid of MT1 to HS-CoM. The activity of MT2 is not limited to the bound methylated corrinoid of MT,; free methylcorrinoids with either 5-hydroxybenzimidazole (HBI) (16) or 5,6-dimethylbenzimidazole (DMBI) as the a-ligand could be demethylated (19, 20). Here we report on the purification and pro...
The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeni was purified 313-fold to a specific activity of 470 ,umol min' mg'1 at 37°C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372Bacteriol. 168: -1377Bacteriol. 168: , 1986, the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barken markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl-and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37°C and pH 7.8. 5,6,7, is the central one-carbon carrier in the intermediate steps of methanogenesis from CO2 (16,17,23,29). In the hydrogenotrophic methanogen Methanobacterium thermoautotrophicum, H4MPT accepts a formyl group from formylmethanofuran and in the formyltransferase reaction 5-formyl-H4MPT is produced (8). The latter compound is converted to 5,10-methenyl-H4MPT ( Fig. 1) by a cyclohydrolase, a monofunctional protein that was purified from the organism (6, 7). After the reduction to 5,10-methylene-H4MPT and 5-methyl-H4MPT and a methyl group transfer, methylcoenzyme M is formed as the substrate of the terminal step of methanogenesis (11,18,20,24). This sequence of H4MPT-dependent reactions bears an analogy to reactions involving tetrahydrofolate derivatives. In fact, H4MPT is a complex structural analog of tetrahydrofolate.When the methylotrophic organism Methanosarcina barkeri is grown on methanol, part of the substrate has to be oxidized to CO2 to obtain the reducing equivalents needed for the methyl group reduction. 5,10-Methenyl-H4MPT was identified as an early intermediate in the substrate conversion (5), suggesting that the oxidative route proceeds via H4MPT derivatives. In this report, the purification and partial characterization of 5,10-methenyl-H4MPT cyclohydrolase from M. barkeri is described. The monofunctional protein catalyzes the hydrolysis of 5,10-methenyl-H4MPT to 10-formyl-H4MPT.MATERIALS AND METHODS Abbreviations. Coenzyme F420, an 8-hydroxy-5-deazaflavin; methanofuran, a 4-substituted-2-(aminomethyl)furan; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TLC, thin-layer chromatography; HPLC, high-performance liquid chromatography.Organism and preparation of cell extracts. M. barkeri MS * Corresponding author.(DSM 800) was grown in a 300-liter fermentor on a synthetic medium containing 250 mM methanol as described previou...
In the process of methanogenesis, 5,6,7,8‐tetrahydromethanopterin (H4MPT) is the carrier of the C1 unit at the formyl through methyl state of reduction. By the transfer of a formyl group from formylmethanofuran, 5‐formyl‐ and 10‐formyl‐H4MPT are formed in hydrogenotrophic and methylotrophic organisms, respectively. Cyclohydrolysis of the 5‐ and 10‐formyl derivatives then yields 5,10‐methenyl‐H4MPT, which is reduced in two subsequent coenzyme F420‐dependent reactions to 5‐methyl‐H4MPT. Following the transfer of the methyl group to coenzyme M, the substrate of the terminal step in methanogenesis, methylcoenzyme M, is produced. In this paper properties of the enzymes catalyzing the individual H4MPT‐dependent reactions are discussed.
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