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2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein

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Abstract

Anaerobically prepared cell-free extracts from Fusobacterium nucleatum contain 2-hydroxyglutaryl-CoA dehydratase with a specific activity of 20 nkat mg-1. The enzyme was purified 24-fold to a specific activity of 480 nkat mg-1 by anion exchange chromatography, gel filtration and chromatography on Blue-Sepharose. The activity of the purified enzyme was strictly dependent on the reductant Ti(III)citrate and stimulated 25-fold by 0.15 mM ATP and 5 mM MgCl2. ATP is hydrolysed to ADP during incubation with 2-hydroxyglutaryl-CoA dehydratase in the presence or absence of the substrate. The enzyme is extremely sensitive towards oxygen and is inhibited by 10 μM chloramphenicol, 10 μM 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits α (49 kDa), β (39 kDa) and γ (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxy-glutaryl-CoA dehydratase from Acidaminococcus fermentans and lactyl-CoA dehydratase from Clostridium propionicum both of which are composed of only two subunits with sizes comparable to those of α and β but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric αβγ-structure. The homogeneous enzyme contains riboflavin (0.5 mol/112 kDa), iron and sulfur (3.5 mol/112 kDa each). Polyclonal antibodies directed against the 2-hydroxyglutaryl-CoA dehydratase from A. fermentans did not crossreact with cell free extracts or purified dehydratase from F. nucleatum. A comparison of the N-terminal amino acid sequences of the dehydratase subunits from A. fermentans and F. nucleatum, however, showed some similarities in the β-subunits.

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Abbreviations

DTT:

dithiothreitol

PAGE:

polyaccrylamide gel electrophoresis

VIS:

visible

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Authors and Affiliations

  1. Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Karl-von-Frisch-Straße, W-3550, Marburg, Federal Republic of Germany

    Anne-Grit Klees & Wolfgang Buckel

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

    Dietmar Linder

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  1. Anne-Grit Klees
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  2. Dietmar Linder
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  3. Wolfgang Buckel
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Klees, AG., Linder, D. & Buckel, W. 2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein. Arch. Microbiol. 158, 294–301 (1992). https://doi.org/10.1007/BF00245248

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  • DOI: https://doi.org/10.1007/BF00245248

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