Critical role of histidine residues in cyclohexanone monooxygenase expression, cofactor binding and catalysis |
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Authors: | Cheesman Matthew J Byron Kneller M Rettie Allan E |
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Institution: | Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA. |
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Abstract: | Cyclohexanone monooxygenase (CMO) is a member of the flavin monooxygenase superfamily of enzymes that catalyze both nucleophilic and electrophilic reactions involving a common C4a hydroperoxide intermediate. To begin to probe structure-function relationships for these enzymes, we investigated the roles of histidine residues in CMO derived from Acinetobacter NCIB 9871, with particular emphasis on the wholly conserved residue, His163 (H163). CMO activity was readily inactivated by diethyl pyrocarbonate (DEPC), a selective chemical modifier of histidine residues. Each of the seven histidines in CMO was then individually mutated to glutamine and the mutants expressed and purified from Escherichia coli. Only the H59Q mutant failed to express at significant levels. The H96Q enzyme was found to have a greatly reduced flavin adenine dinucleotide (FAD) content, indicative of compromised cofactor retention. The only significant effect on kcat occurred with the H163Q mutant, which exhibited an approximately 10-fold lower turnover of the prototypical substrate, cyclohexanone. This was accompanied by a doubling in the Km NADPH] compared to the wild-type enzyme, suggesting that the functional decrement in H163Q is probably not solely a reflection of impaired NADPH binding. These data establish a critical role for H163 in CMO catalysis and prompt the hypothesis that this conserved residue plays a similarly important functional role across the flavin monooxygenase family of enzymes. |
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Keywords: | CMO cyclohexanone monooxygenase FMO flavin-containing monooxygenase DEPC diethyl pyrocarbonate IPTG d-thiogalactopyranoside" target="_blank">isopropyl-β-d-thiogalactopyranoside FAD flavin adenine dinucleotide CHCA α-cyano-4-hydroxycinnamic acid PCR polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis MeCN acetonitrile TFA trifluoroacetic acid MALDI matrix-assisted laser desorption ionization LC-MS liquid chromatography-mass spectrometry HPLC high performance liquid chromatography ESI-MS electrospray ionization-mass spectrometry |
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