On the enzymatic mechanism of 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile

The genes ldhA and hadA, from Clostridium difficile (DSMZ 1296T) were cloned and expressed in Escherichia coli. The obtained proteins were purified and characterised as D-2-hydroxyisocaproate dehydrogenase (LdhA) and 2-hydroxyisocaproate CoA-transferase (HadA) involved in two consecutive steps in the pathway of leucine fermentation to ammonia, CO2, isovalerate and isocaproate. The downstream genes hadBC and hadI were also functionally expressed and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase (HadBC) and its activator (HadI). The activated dehydratase catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA, which is a chemically difficult step since the proton in the b-position is not activated (pK ca. 40). We postulated that the reduction of the substrate by one electron enables the elimination, whereby the pK is lowered to at least 14. After the reaction the electron is returned to the dehydratase, which may catalyse many turnovers. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase contain iron-sulfur cluster(s); other prosthetic groups specifically molybdenum were not detected. The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase. The reduced dehydratase separated from the activator and ATP catalysed almost 104 dehydration turnovers until the electron was lost by oxidation. By adding activator and ATP the enzyme could be fully reactivated. The active tight complex of the two protein components induced by AlF4- and ATP underpins the postulated electron transfer from the activator to the dehydratase and demonstrates again that the electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (activator/dehydratase = 1:10) were required to generate full activity. An organic radical proposed to mediate the dehydration was detected by EPR spectroscopy for the first time in a 2-hydroxyacyl-CoA dehydratase. The changes of the EPR spectra induced by the use of labelled substrates showed that the radical was substrate-derived. These results are the first clear evidence for a radical involved in a dehydration mechanism and suggest a new way to form a radical in enzymatic reactions.