2‐Hydroxyisocaproyl‐CoA dehydratase and its activator from Clostridium difficile

Abstract

The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI. The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation. The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase. 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 thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive. Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme. This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover. In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.

Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction ofl-Leucine to Isocaproate byClostridium difficile

“…Since synthetic (E)-2-isocaprenoyl-CoA as well as the CoA ester prepared with the CoA transferase also were not hydrated by the dehydratase (20), we conclude that the thereby unavoidable treatments with acid caused an unknown change in the molecule.…”

Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes

“…Two gene clusters that encode key enzymes in the fermentation pathways of these two amino acids were identified in the C. botulinum genome. The first system, CBO2192-2199, is similar to 2-hydroxyisocaproyl-CoA dehydratase (HadAIBC) from C. difficile, in the pathway of leucine fermentation (Kim et al 2005). The second, CBO3283-3292, which is missing in the other sequenced clostridial genomes, is highly similar to phenyllactate dehydratase (FldAIBC) from C. sporogenes, which is involved in the fermentation of phenylalanine (Dickert et al 2002).…”

Sodium Ion Pumps and Hydrogen Production in Glutamate Fermenting Anaerobic Bacteria

“…It has been measured with the related 2-hydroxyisocaproyl-CoA dehydratase from Clostridium diffi cile that this single electron participates in about 10,000 turnovers until it is lost by oxidation [Kim et al, 2005]. ATP-driven electron transfer reactivates the now inactive dehydratase by injecting a new electron.…”