N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase (H₂-forming) from the extreme thermophile Methanopyrus kandleri

Abstract

Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110°C on H₂ and CO₂. The N⁵, N¹⁰-methylenetetrahydromethanopterin dehydrogenase, an enzyme involved in methanogenesis from CO₂ and H₂, was purified from this hyperthermophile and characterized. The dehydrogenase was found to be composed of only one polypeptide of apparent molecular mass 44 kDa. The UV/Vis spectrum was similar to that of albumin. The protein catalyzed the reversible dehydrogenation of N⁵, N¹⁰-methylenetetra-hydromethanopterin (CH₂=H₄MPT) to N⁵, N¹⁰-methenyltetrahydromethanopterin (CH = H₄MPT⁴) and molecular hydrogen: CH = H₄MPT⁴ + H₂. The rate of CH₂=H₄MPT dehydrogenation (apparent V_max) at 65°C and pH5.8 was 1500 U/mg, the apparent K_m for CH₂=H₄MPT was 50 μM, the Arrhenius activation energy was 52 kJ/mol, and the Q₁₀ between 30°C and 70°C was 2.-. The specific activity increased hyperbolically with the proton concentration between pH 7 and pH 4.5. The purified dehydrogenase did not catalyze the reduction of viologen dyes, of coenzyme F₄₂₀, and of pyridine nucleotides with either CH₂=H₄MPT or H₂. For activity the CH₂=H₄MPT dehydrogenase required the presence of salts. Fifty percent of maximal activity was reached at salt concentrations of 100 mM, potassium phosphate, potassium chloride, and sodium chloride being almost equally effective in stimulating the enzyme activity. Cell extracts of M. kandleri did not loose CH₂=H₄MPT dehydrogenase activity when incubated at 90°C for 60 min. The purified enzyme, however, proved very themolabile. The N-terminal amino acid sequence of the dehydrogenase was determined and compared with that of the CH₂=H₄MPT dehydrogenase (H₂-forming) from Methanobacterium thermoautotrophicum. Significant similarity was found.