Isolation of a 5-hydroxybenzimidazolyl cobamide-containing enzyme involved in the methyltetrahydromethanopterin: coenzyme M methyltransferase reaction in Methanobacterium thermoautotrophicum.

Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597).

Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri.

The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees 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. 168:1372-1377, 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. barkeri 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 degrees C and pH 7.8.

Identification of 6-acetyl-7-methyl-7,8-dihydropterin as a degradation product of 5,10-methenyl-5,6,7,8-tetrahydromethanopterin.

During purification procedures and upon aerobic heating with alkali a green-yellow degradation fluorescent product (GY) was formed from 5,10-methenyl-5,6,7,8-tetrahydromethanopterin, an intermediate in the reduction of CO2 to methane [J. T. Keltjens, L. Daniels, H. G. Janssen, and G. D. Vogels (1983) Eur. J. Biochem. 130, 545-552]. GY was suggested to be a 6-(1-oxo)-7,8-dihydropterin. On the basis of the spectral properties and the results of degradation studies, it was now shown that the structure of GY is 6-acetyl-7-methyl-7,8-dihydropterin. This structure was confirmed by synthesis of the compound and other reference substances.

Structural elements of methanopterin, a novel pterin present in Methanobacterium thermoautotrophicum.

During short-time labeling experiments, cells of Methanobacterium thermoautotrophicum incorporate a substantial part of 14CO2 in a yellow fluorescent compound (called YFC) [Daniels, L. & Zeikus, J. G. (1978) J. Bacteriol. 136, 75-84]. As the compound was present only in small amounts, its more abundant, metabolic precursor was identified, extracted and purified by column chromatography. The chromophore of this compound is 2-amino-4-hydroxypteridine (pterin) as indicated by its ultraviolet-visible-light absorption and fluorescence properties. Decomposition studies revealed the presence of a number of structural elements, viz. glutamic acid, phosphate and a hexosamine. 1H-NMR and 13C-NMR spectra pointed to the presence of additional, as yet unidentified, elements. The compound is a complex, novel pterin derivative, which we have called methanopterin.

A novel one-carbon carrier (carboxy-5,6,7,8-tetrahydromethanopterin) isolated from Methanobacterium thermoautotrophicum and derived from methanopterin.

During short-term labeling experiments, cells of Methanobacterium thermoautotrophicum incorporated a substantial part of 14CO2 in a compound with a bright yellow fluorescence on dry thin-layer chromatography plates and called yellow fluorescent compound (YFC) [Daniels, L. and Zeikus, J.G. (1978) J. Bacteriol. 136, 75-84]. This compound was extracted and purified by ion-exchange column chromatography with formic acid gradients up to 0.3 M. Out of 325 g wet cells of M. thermoautotrophicum about 4 mg of the compound were isolated. This material and some degradation products obtained from it were studied by means of chemical decomposition, ultraviolet-visible-light spectroscopy and preliminary 1H-NMR spectroscopy. It has structural elements in common with methanopterin (see preceding paper in this journal); these elements are a pterin group, glutamate, a hexosamine. The pterin in this compound is present in a reduced form, presumably as 5,6,7,8-tetrahydromethanopterin, and the additional one-carbon unit is probably present as a carboxy group. Probably the first step of methanogenesis implies a carboxylation of methanopterin and a concomitant reduction of the pterin. The trivial name carboxy-5,6,7,8-tetrahydromethanopterin is introduced for the compound.