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Abstract

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogens known so far. N5N10-Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F420 as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. Vmax at 65°C and pH 6.8 was 435 U/mg (kcat=275 s-1) and the Km for methylenetetrahydro-methanopterin and for reduced F420 were 6 μM and 4 μM, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q10 between 40°C and 90°C was 1.5.