Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

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Abstract
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 210000) consisting of two different polypeptides (α, Mr 55000, and β, Mr 42000) in an α2β2 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 A°. 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, 13181–13189].