Dehydration of ( R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of α-amino acids by anaerobic bacteria

Abstract

Several clostridia and fusobacteria ferment α-amino acids via ( R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination. This reaction is of great mechanistic interest, since the β-hydrogen, to be eliminated as proton, is not activated (p K 40–50). A mechanism has been proposed, in which one high-energy electron acts as cofactor and transiently reduces the electrophilic thiol ester carbonyl to a nucleophilic ketyl radical anion. The 2-hydroxyacyl-CoA dehydratases are two-component systems composed of an extremely oxygen-sensitive component A, an activator, and component D, the actual dehydratase. Component A, a homodimer with one [4Fe–4S]cluster, transfers an electron to component D, a heterodimer with 1-2 [4Fe–4S]clusters and FMN, concomitant with hydrolysis of two ATP. From component D the electron is further transferred to the substrate, where it facilitates elimination of the hydroxyl group. In the resulting enoxyradical the β-hydrogen is activated (p K14). After elimination the electron is handed-over to the next incoming substrate without further hydrolysis of ATP. The helix–cluster–helix architecture of component A forms an angle of 105°, which probably opens to 180° upon binding of ATP resembling an archer shooting arrows. Therefore we designated component A as `Archerase'. Here, we describe 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, 2-phenyllactate dehydratase from Clostridium sporogenes, 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, and lactyl-CoA dehydratase from Clostridium propionicum. A relative of the 2-hydroxyacyl-CoA dehydratases is benzoyl-CoA reductase from Thauera aromatica. Analogous but unrelated archerases are the iron proteins of nitrogenase and bacterial protochlorophyllide reductase. In anaerobic organisms, which do not oxidize 2-oxo acids, a second energy-driven electron transfer from NADH to ferredoxin, the electron donor of component A, has been established. The transfer is catalysed by a membrane-bound NADH–ferredoxin oxidoreductase driven by an electrochemical Na +-gradient. This enzyme is related to the Rnf proteins involved in Rhodobacter capsulatus nitrogen fixation.