title: Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide.
abstract: Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan.
authors.forename: Qing-Shan
authors.initials: QS
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Ogawa
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
mesh_terms: D000494:Allosteric Regulation; D001410:Bacillus megaterium / Q000201:enzymology / Q000235:genetics; D001555:Benzene Derivatives / Q000737:chemistry*; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007211:Indoles / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D017354:Point Mutation
chemical_list: D001555:Benzene Derivatives; D007211:Indoles; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C007164:cumene hydroperoxide