Critical role of the residue size at position 87 in H2O2- dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3.
Replacement of phenylalanine 87 with alanine or glycine (mutant F87A or F87G) greatly increased the H2O2-supported substrate hydroxylation activity of cytochrome P450BM-3, whose original H2O2-supported activity is hardly detectable. On the other hand, replacement of phenylalanine 87 with valine (mutant F87V) did not. In the oxidation of p-nitrophenoxydodecanoic acid (12-pNCA), the turnover numbers of the mutant F87A in the presence of NADPH and O2, or H2O2 were 493 and 162 nmol/min/nmol, respectively. The H2O2-supported F87A hydroxylation activity was further confirmed with free fatty acids as substrates. Moreover, the stability of F87A in H2O2 solutions also largely increased. The order of the stability of the wild type (WT), F87A, and their substrate (12-pNCA)-binding complexes in H2O2 solutions listed from high to low was F87A, WT, F87A substrate-binding complex, and WT substrate-binding complex. We propose that the free space size in the vicinity of the heme iron significantly influences P450BM-3 H2O2-supported activity and H2O2 inactivation.
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Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.
Ogawa
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J
Shimizu
S
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D019943:Amino Acid Substitution / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005227:Fatty Acids / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000494:pharmacology; D009251:NADPH-Ferrihemoprotein Reductase; D010649:Phenylalanine / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D010649:Phenylalanine; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases