2-Hydroxyglutaryl-CoA dehydratase from Fusobacterium nucleatum (subsp. nucleatum): an iron-sulfur flavoprotein.
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 microM chloramphenicol, 10 microM 2,4-dinitrophenol or 0.15 mM hydroxylamine. The pure enzyme consists of three subunits alpha (49 kDa), beta (39 kDa) and gamma (24 kDa) in approximately equal amounts. In this respect the enzyme differs from the related 2-hydroxyglutaryl-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 alpha and beta but require an additional protein for activity. The relative molecular mass of the native enzyme of about 100 kDa suggests a trimeric alpha beta gamma-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 beta-subunits.
Klees
A G
AG
Laboratorium für Mikrobiologie des Fachbereichs Biologie, Philipps-Universität, Marburg, Federal Republic of Germany.
Linder
D
D
Buckel
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D005285:Fermentation; D005420:Flavoproteins / Q000378:metabolism; D005609:Free Radicals / Q000032:analysis; D016967:Fusobacterium nucleatum / Q000201:enzymology*; D006836:Hydro-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008969:Molecular Sequence Data
D005420:Flavoproteins; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D000255:Adenosine Triphosphate; C054464:2-hydroxyglutaryl-CoA dehydratase; D006836:Hydro-Lyases