N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus |
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Authors: | R. A. Schmitz D. Linder K. O. Stetter R. K. Thauer |
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Affiliation: | (1) Laboratorium für Mikrobiologie, FB Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, W-3550 Marburg, Federal Republic of Germany;(2) Fachbereich Humanmedizin, Biochemisches Institut, Justus-Liebig-Universität Giessen, W-6300 Giessen, Federal Republic of Germany;(3) Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, W-8400 Regensburg, Federal Republic of Germany |
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Abstract: | Methylene-H4MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F420 as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a Km for methylene-H4MPT of 16 M, a Km for F420H2 of 4 M, and a Vmax of 450 U/mg (Kcat=265 s-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F420 (0.2 mM), methylene-H4MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H4MPT reductase (F420-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H4MPT tetrahydromethanopterin - CH2=H4MPT N5,N10-methylene-H4MPT - CH3–H4MPT N5-methyl-H4MPT - CH H4MPT methenyl-H4MPT - F420 coenzyme F420 - MFR methanofuran - CHO-MFR formyl-MFR - 1 U 1 mol/min |
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Keywords: | Sulfate-reducing archaebacteria Hyperthermophilic bacteria Archaeglobus fulgidus Tetrahydromethanopterin Methanofuran Coenzyme F420 Thermostable enzymes |
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