Activation of (R)-2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans.

(R)-2-Hydroxyglutaryl-CoA dehydratase (HgdAB) from Acidaminococcus fermentans catalyses the reversible dehydration of its substrate to glutaconyl-CoA. The enzyme has to be activated by ATP, MgCl2, and Ti(III)citrate by an activator protein (HgdC) that is present in the organism at very low concentrations. Cell-free extracts of a recombinant Escherichia coli strain, in which hgdC was expressed, contained the activator with a specific activity of up to 45 U'/mg protein (1 U' is the amount of activator required to generate 1 U dehydratase activity under standard assay conditions). The recombinant protein was purified 44-fold to a specific activity of 2000 U'/mg. It is a homodimer (gamma 2, 54 kDa) and contains 4 mol non-heme iron and 3 mol inorganic sulfur. Under air, the activator has a half-life of seconds and even under strict anaerobic conditions it is very unstable. The amino acid sequence of the activator shows similarities to the ATP-binding motifs of several kinases. The dehydratase component was purified from its natural source revealing a heterodimer (alpha beta, 100 kDa) that contains 4 mol non-heme iron, 4 mol inorganic sulfur, 0.3 mol riboflavin, and 1 mol FMN. A mechanism is proposed in which an iron-sulfur cluster or a flavin donates one electron to the thiolester of the substrate (R)-2-hydroxyglutaryl-CoA. The resulting ketyl may eliminate the adjacent hydroxyl group yielding an enoxy radical from which the beta-hydrogen is abstracted as a proton leading to the ketyl of glutaconyl-CoA. In the final step, the latter is oxidized to the product, whereby the reduced enzyme is regenerated. It is suggested that during the activation step, the electron of this cycle is fed into the enzyme by Ti(III)citrate and energized by hydrolysis of ATP; both functions are apparently catalysed by the activator. The enzyme remains in this activated state for several turnovers, which may explain the requirement of only catalytic amounts of ATP and substoichiometric amounts of activator (dehydratase/activator ratio approximately 200:1). The oxidants 4-nitrobenzoate, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone or chloramphenicol (all at concentrations greater than or equal to 1 microM) may trap this electron resulting in a reversible, transient inactivation of the dehydratase.