title: 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.
authors.affiliation: Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, 35032 Marburg, Germany.
authors.forename: Jihoe
authors.initials: J
authors.lastname: Kim
authors.forename: Daniel
authors.initials: D
authors.lastname: Darley
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Buckel
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000201:enzymology*; D004789:Enzyme Activation; D005285:Fermentation; D007930:Leucine / Q000378:metabolism*
chemical_list: D015094:3-Hydroxyacyl CoA Dehydrogenases; D000251:Adenosine Triphosphatases; D007930:Leucine