Salt dependence, kinetic properties and catalytic mechanism of N-formylmethanofuran:tetrahydromethanopterin formyltransferase from the extreme thermophile Methanopyrus kandleri

Authors

  • Jürgen BREITUNG,

    1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität and Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Federal Republic of Germany
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  • Gerhard BÖRNER,

    1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität and Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Federal Republic of Germany
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  • Sabine SCHOLZ,

    1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität and Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Federal Republic of Germany
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  • Dietmar LINDER,

    1. Fachbereich Humanmedizin, Biochemisches Institut, Justus-Liebig-Universität Giessen, Federal Republic of Germany
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  • Karl O. STETTER,

    1. Lehrstuhl für Mikrobiologie, Universität Regensburg, Federal Republic of Germany
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  • Rudolf K. THAUER

    Corresponding author
    1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität and Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Federal Republic of Germany
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Correspondence to R. K. Thauer, Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karl-von-Frisch-Strasse, W-3550 Marburg, Federal Republic of Germany
Fax: +49 6421 285833.

Abstract

N-Formylmethanofuran(CHO-MFR): tetrahydromethanopterin(H4MPT) formyltransferase (for-myltransferase) from the extremely thermophilic Methanopyrus kandleri was purified over 100-fold to apparent homogeneity with a 54% yield. The monomeric enzyme had an apparent molecular mass of 35 kDa. The N-terminal amino acid sequence of the polypeptide was determined.

The formyltransferase was found to be absolutely dependent on the presence of phosphate or sulfate salts for activity. The ability of salts to activate the enzyme decreased in the order K2HPO4 > (NH4)2SO4 > K2SO4 > Na2SO4 > Na2HPO4. The salts KCl, NaCl and NH4Cl did not activate the enzyme. The dependence of activity on salt concentration showed a sigmoidal curve. For half-maximal activity, 1 M K2HPO4 and 1.2 M (NH4)2SO4 were required. A detailed kinetic analysis revcaled that phosphates and sulfates both affected the Vmax rather than the Km for CHO-MFR and H4MPT. At the optimal salt concentration and at 65°C, the Vmax was 2700 U/mg (1 U = 1 μmol/min), the Km for CHO-MFR was 50 μM and the Km for H4MPT was 100 μM. At 90°C, the temperature optimum of the enzyme, the Vmax was about 2.5-fold higher than at 65°C.

Thermostability as well as activity of formyltransferase was dramatically increased in the presence of salts, 1.5 M being required for optimal stabilization. The efficiency of salts in protecting formyltransferase from heat inactivation at 90°C decreased in the order K2HPO4= (NH4)2SO4≫ KCI = NH4Cl = NaCl ≫ Na2SO4 > Na2HPO4. The catalytic mechanism of formyltransferase was determined to be of the ternary-complex type. The properties of the enzyme from M. kandleri are compared with those of formyltransferase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri and Archaeoglobus fulgidus.

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