TAILIEUCHUNG - Báo cáo khoa học: 2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile

ThehadBC and hadI genes from Clostridium difficilewere functionally expressed inEscherichia coliand shown to encode the novel 2-hydroxyiso-caproyl-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 dehy-dratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehy-dratase | iFEBS Journal 2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile Jihoe Kim Daniel Darley and Wolfgang Buckel Laboratorium fur Mikrobiologie Fachbereich Biologie Philipps-Universitat Marburg Germany Keywords ATP iron-sulfur leucine fermentation electron recycling radical mechanism Correspondence W. Buckel Laboratorium for Mikrobiologie Fachbereich Biologie Philipps-Universitat 35032 Marburg Germany Fax 49 6421 28 28979 Tel 49 6421 28 21527 E-mail buckel@ Received 4 August 2004 revised 12 November 2004 accepted 22 November 2004 doi The hadBC and hadl genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyiso-caproyl-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 104 dehydration turnovers until the enzyme became inactive. Adding activator ATP MgCl2 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. 2-Hydroxyacyl-CoA dehydratases are the key enzymes in the fermentation pathways of 12 proteinogenous amino acids to ammonia

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