title: Two N5,N10-methylenetetrahydromethanopterin dehydrogenases in the extreme thermophile Methanopyrus kandleri: characterization of the coenzyme F420-dependent enzyme.
abstract: It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N5,N10-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F420-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7 +/- 0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH4)2SO4, 2 M Na2HPO4, 1.5 M K2HPO4, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the Vmax rather than the Km of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of 1 M (NH4)2SO4 the Vmax was 4000 U/mg (kcat = 2400 s-1) and the Km for N5,N10-methylenetetrahydromethanopterin and for coenzyme F420 were 80 microM and 20 microM, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K2HPO4 at 90 degrees C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.
authors.affiliation: Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg, Germany.
authors.forename: A R
authors.initials: AR
authors.lastname: Klein
authors.forename: J
authors.initials: J
authors.lastname: Koch
authors.forename: K O
authors.initials: KO
authors.lastname: Stetter
authors.forename: R K
authors.initials: RK
authors.lastname: Thauer
mesh_terms: D000595:Amino Acid Sequence; D019605:Euryarchaeota / Q000201:enzymology* / Q000235:genetics; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012256:Riboflavin / Q000031:analogs & derivatives* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: C015084:coenzyme F420; D000587:Oxidoreductases Acting on CH-NH Group Donors; C047126:methylenetetrahydromethanopterin dehydrogenase; D012256:Riboflavin