title: Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450.
abstract: CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.
authors.affiliation: Department of Virology, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan. matsunagai@med.osaka-cu.ac.jp
authors.forename: I
authors.initials: I
authors.lastname: Matsunaga
authors.forename: A
authors.initials: A
authors.lastname: Ueda
authors.forename: T
authors.initials: T
authors.lastname: Sumimoto
authors.forename: K
authors.initials: K
authors.lastname: Ichihara
authors.forename: M
authors.initials: M
authors.lastname: Ayata
authors.forename: H
authors.initials: H
authors.lastname: Ogura
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D019814:Myristic Acid / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D019814:Myristic Acid; D013912:Threonine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D011392:Proline; D006861:Hydrogen Peroxide; D010544:Peroxidases; C066418:cytochrome P-450 peroxygenase; C437763:cytochrome P-450 CYP152A1 (Bacillus subtilis); D008239:Lysine; D009584:Nitrogen