Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein.
1. The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose. It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two [4Fe-4S] centers. After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. 2. The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as Ao. Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol. 3. The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13,181-13,189].
Biochemie I, Universität Regensburg, Federal Republic of Germany.
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Schweiger
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Dutscho
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Buckel
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology*; D006836:Hydro-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008982:Molybdenum / Q000032:analysis; D013455:Sulfur / Q000032:analysis
D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D013455:Sulfur; D008982:Molybdenum; D007501:Iron; C054464:2-hydroxyglutaryl-CoA dehydratase; D006836:Hydro-Lyases