tailieunhanh - Báo cáo khoa học: Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii Implications for the disulfide metabolism of anaerobic hyperthermophiles

We have cloned NADH oxidase homologues fromPyrococcus horikoshii and P. furiosus, and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes (previously referred to as NOX2) have been shown to act as a coenzyme A disulfide reductases (CoADR: CoA-S-S-CoA + NAD(P)H + H +fi2CoA-SH + NAD(P) + ). The P. horikoshiienzyme shows a kcat app of s )1 with NADPH at 75 C. | ềFEBS Journal Discovery and characterization of a Coenzyme A disulfide reductase from Pyrococcus horikoshii Implications for the disulfide metabolism of anaerobic hyperthermophiles Dennis R. Harris Donald E. Ward1 Jeremy M. Feasel2 Kyle M. Lancaster2 Ryan D. Murphy2 T. Conn Mallet3 and Edward J. Crane III2 1 Genencor International Palo Alto CA USA 2 Department of Chemistry Pomona College Claremont CA USA 3 Center for StructuralBiology Wake Forest University Schoolof Medicine Winston-Salem NC USA Correspondence E. J. Crane III Department of Chemistry Pomona College 645 North College Avenue Claremont CA 91711 USA Fax 1 909 607 7726 Tel 1 909 607 9634 E-mail Website http . ejc14747 Present address Department of Biochemistry University of Wisconsin-Madison Madison WI USA Received 20 July 2004 revised 7 December 2004 accepted 4 January 2005 doi We have cloned NADH oxidase homologues from Pyrococcus horikoshii and P. furiosus and purified the recombinant form of the P. horikoshii enzyme to homogeneity from Escherichia coli. Both enzymes previously referred to as NOX2 have been shown to act as a coenzyme A disulfide reductases CoADR CoA-S-S-CoA NAD P H H fi 2CoA-SH NAD P . The P. horikoshii enzyme shows a kcatapp of s-1 with NADPH at 75 C. While the enzyme shows a preference for NADPH it is able to use both NADPH and NADH efficiently with both giving roughly equal kcats while the Km for NADPH is roughly eightfold lower than that for NADH. The enzyme is specific for the CoA disulfide and does not show significant reductase activity with other disulfides including dephos-pho-CoA. Anaerobic reductive titration of the enzyme with NAD P H proceeds in two stages with an apparent initial reduction of a nonflavin redox center with the first reduction resulting in what appears to be an EH2 form of the enzyme. Addition of a second of NADPH results in the formation of an apparent FAD-NAD P H .

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