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N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus

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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 K m for methylene-H4MPT of 16 μM, a K m for F420H2 of 4 μM, and a V max 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.

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Abbreviations

H4MPT:

tetrahydromethanopterin

CH2=H4MPT:

N 5,N 10-methylene-H4MPT

CH3−H4MPT:

N 5-methyl-H4MPT

CH≡H4MPT:

methenyl-H4MPT

F420 :

coenzyme F420

MFR:

methanofuran

CHO-MFR:

formyl-MFR

1 U:

1 μmol/min

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Authors and Affiliations

  1. Laboratorium für Mikrobiologie, FB Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, W-3550, Marburg, Federal Republic of Germany

    R. A. Schmitz & R. K. Thauer

  2. Fachbereich Humanmedizin, Biochemisches Institut, Justus-Liebig-Universität Giessen, W-6300, Giessen, Federal Republic of Germany

    D. Linder

  3. Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, W-8400, Regensburg, Federal Republic of Germany

    K. O. Stetter

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  1. R. A. Schmitz
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  2. D. Linder
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  3. K. O. Stetter
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  4. R. K. Thauer
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Schmitz, R.A., Linder, D., Stetter, K.O. et al. N 5,N 10-Methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) and formylmethanofuran dehydrogenase from the hyperthermophile Archaeoglobus fulgidus . Arch. Microbiol. 156, 427–434 (1991). https://doi.org/10.1007/BF00248722

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