N5, N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming) from the extreme thermophile Methanopyrus kandleri.
Methanopyrus kandleri is a novel abyssal methanogenic archaebacterium growing at 110 degrees 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-methylenetetrahydromethanopterin (CH2 = H4MPT) to N5, N10-methenyltetrahydromethanopterin (CH identical to H4MPT+) and molecular hydrogen: CH2 = H4MPT H+ in equilibrium CH identical to H4MPT+ +H2. The rate of CH2 = H4MPT dehydrogenation (apparent Vmax) at 65 degrees C and pH 5.8 was 1500 U/mg, the apparent Km for CH2 = H4MPT was 50 microM, 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 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 degrees C for 60 min. The purified enzyme, however, proved very thermolabile. The purified enzyme, however, proved very thermolabile.(ABSTRACT TRUNCATED AT 250 WORDS)
Ma
K
K
Laboratorium für Mikrobiologie, Fachbereich Biologie, Phillips-Universität Marburg, Marburg/Lahn, Federal Republic of Germany.
Zirngibl
C
C
Linder
D
D
Stetter
K O
KO
Thauer
R K
RK
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D019605:Euryarchaeota / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D012965:Sodium Chloride; D011189:Potassium Chloride; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase