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The genes coding for methyl coenzyme M reductase were cloned from a genomic library ofMethanobacterium thermoautotrophicum Marburg into Escherichia coli by using plasmid expression vectors. When introduced into E. coli, the reductase genes were expressed, yielding polypeptides identical in size to the three known subunits of the isolated enzyme, a, 13, and y. The polypeptides also reacted with the antibodies raised against the respective enzyme subunits. In M. thermoautotrophicum, the subunits are encoded by a gene cluster whose transcript boundaries were mapped. Sequence analysis revealed two more open reading frames of unknown function located between two of the methyl coenzyme M reductase genes.Methanogenic bacteria are archaebacteria, which gain their energy by anaerobic formation of methane (6). Independent of the methanogenic substrate, the final step of methanogenesis is the reduction of methyl coenzyme M (CoM) to methane and CoM. This reaction, which is coupled to the synthesis of ATP in vivo (7,20), is catalyzed by the methyl CoM reductase (MCR) (3,12). This enzyme amounts to about 10% of the total protein (3, 12) and has been isolated from a large number of methanogenic bacteria (3,18,21 Q359 (19) hsdR supE tonA (P2) Y1089 (42) AlacUl69 Alon araDI39 strA hflA150 chr::TnlO (pMC9) Y1090 (42) A1acUl69 Alon araD139 strA supF trpC22::TnlO (pMC9) BNN97 (41) hsdR supE thr leu thi lacYI tonA21 (Xgtll) pUC8 (26, 39) XEMBL4 (13) Agtll (37) previously (23), using formaldehyde gels. Transfer of the DNA to GeneScreen sheets essentially followed the procedure described by Thomas (37) with the modifications given in the GeneScreen manual by the supplier. The DNA probe was labeled by nick translation as described in reference 24, using a-32P-labeled deoxynucleoside triphosphates to a specific activity of 0.5 x 108 to 1 x 108 cpm/,Lg of DNA. Hybridization and further processing of the filters were performed as described in reference 15.Antisera against MCR subunits. Antisera were raised in rabbits against the subunits of purified MCR, which were eluted from an SDS-polyacrylamide gel after their electrophoretic separation. The specificities were checked in Western blot (immunoblot) experiments, using total extracts of E. coli or M. thermoautotrophicum cells or purified MCR. No reactions were observed with E. coli extracts. The antisera were found to react with one polypeptide band each in the M. thermoautotrophicum extract, which corresponded in size to the respective subunit reacting with the same antiserum.Screening of M. thermoautotrophicum genomic libraries. Two DNA libraries were constructed. First, an EcoRI total digest of M. thermoautotrophicum DNA was ligated to Agtll DNA, which was digested with EcoRI and treated with calf intestinal phosphatase (41). After in vitro packaging (42), bacteriophages were plated on E. coli Y1090 and screened with antisera against the isolated subunits after induction with isopropyl-1-D-thiogalactopyranoside as described below. Positive phages were lysogenized in E. coli Y1089 an...

H,-forming N',N"'-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N"'-methenyltetrahydomethanopterin (CH=H,MPT.') with H, to N',N"'-methylenetetrahydromethanopterin (CH,=H,MPT) and a proton (AGO' = -5.5 kJ/mol). The enzyme also catalyzes a CH-H,MPT+-dependent H,/H' exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH=H,MPT' reduction was performed with DZ instead of H2, V,,,., and the K,,, did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H,O or DZO was the solvent. The findings indicate that a step other than the activation of H, was rate-determining in CH=H,MPT+ reduction with H,. This was substantiated by the observation that also the CH=H,MPT+-dependent H,/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. V,,,,, for CH,=H,MPT dehydrogenation to CH-H,MPT+ and H2 was only 2-3 times higher than for CD2=H,MPT dehydrogenation to CD-H,MPT' and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2=H,MPT was only rate-limiting when hydrogen was substituted by a deuterium.

The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.

The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N5,N10-methylenetetrahydromethanopterin dehydrogenase (coenzyme F420 dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity.

Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83 degrees C and 98 degrees C, respectively. Both Archaea contain an active N5,N10-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus. The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K2HPO4 and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K2HPO4 (1 M) for optimal thermostability at 90 degrees C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K2HPO4 concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.

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Coenzyme F,,,-dependent methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea catalyzes the reversible transfer of a hydride ion from C14a of N5,Mo-methylenetetrahydromethanopterin to C5 of coenzyme F,,,. In this study, we report that this hydride transfer proceeds stereospecifically from the Re face at C14a to the Si face at C5. The results were obtained by using chirally 3H-labelled N5,Mo-methylenetetrahydromethanopterin generated via Re-face-specific H,-forming N5,Wo-methylenetetrahydromethanopterin dehydrogenase and by analyzing reduced coenzyme F,,, via Si-face-specific F,,,-reducing hydrogenase.Keywords. Coenzyme F,,, ; stereospecificity ; methylenetetrahydromethanopterin dehydrogenase ; hydrogenase.Coenzyme F4,,-dependent methylene tetrahydromethanopterin dehydrogenase catalyzes the reversible conversion of N5,Mo-methylenetetrahydromethanopterin (CH,=H,MPT) and coenzyme F,,, (F,,,) to W,Mo-methenyl-H,MPT (CH= H4MPT+) and reduced coenzyme F,,, (F4zoH,) (reaction a; for structures of CH,=H,MPT, CH=KMPT+, F,,, and F42, HZ see The enzyme is found in methanogenic Archaea (Schworer and Thauer, 1991) and Archaeoglobus species (Moller-Zinkhan et al., 1989;Vorholt et al., 1994 on lactate plus sulfate, the enzyme catalyzes the oxidation of CH,=H,MPT to CH-H,MPT+ as an intermediary step in CO, formation (Keltjens and Vogels, 1993; Thauer and Kunow, 1995).