N5, N-10-METHYLENETETRAHYDROMETHANOPTERIN DEHYDROGENASE (H-2-FORMING) FROM THE EXTREME THERMOPHILE METHANOPYRUS-KANDLERI

Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110-degrees-C on H-2 and CO2. The N5,N-10-methylenetetrahydromethanopterin dehydrogenase, an enzyme involved in methanogenesis from CO2 and H-2, 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,N-10-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5,N-10-methenyltetrahydromethanopterin (CH = H4MPT+) and molecular hydrogen: CH2 = H4MPT + H+ reversible CH = H4MPT+ + H-2. The rate of CH2 = H4MPT dehydrogenation (apparent V(max)) at 65-degrees-C and pH 5.8 was 1500 U/mg, the apparent K(m) for CH2 = H4MPT was 50-mu-M, the Arrhenius activation energy was 52 kJ/mol, and the Q10 between 30-degrees-C and 70-degrees-C was 2.0. 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 H-2. 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-degrees-C for 60 min. The purified enzyme, however, proved very thermolabile. The N-terminal amino acid sequence of the dehydrogenase was determined and compared with that of the CH2 = H4MPT dehydrogenase (H-2-forming) from Methanobacterium thermoautotrophicum. Significant similarity was found.