title: Rational engineering of the fungal P450 monooxygenase CYP5136A3 to improve its oxidizing activity toward polycyclic aromatic hydrocarbons.
abstract: A promising polycyclic aromatic hydrocarbon-oxidizing P450 CYP5136A3 from Phanerochaete chrysosporium was rationally engineered to enhance its catalytic activity. The residues W129 and L324 found to be critical in substrate recognition were transformed by single (L324F) and double (W129L/L324G, W129L/L324F, W129A/L324G, W129F/L324G and W129F/L324F) mutations, and the engineered enzyme forms were expressed in Pichia pastoris. L324F and W129F/L324F mutations enhanced the oxidation activity toward pyrene and phenanthrene. L324F also altered the regio-selectivity favoring C position 4 over 9 for hydroxylation of phenanthrene. This is the first instance of engineering a eukaryotic P450 for enhanced oxidation of these fused-ring hydrocarbons.
authors.affiliation: Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0056, USA.
authors.forename: Khajamohiddin
authors.initials: K
authors.lastname: Syed
authors.forename: Aleksey
authors.initials: A
authors.lastname: Porollo
authors.forename: David
authors.initials: D
authors.lastname: Miller
authors.forename: Jagjit S
authors.initials: JS
authors.lastname: Yadav
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D020075:Phanerochaete / Q000737:chemistry* / Q000201:enzymology; D010616:Phenanthrenes / Q000737:chemistry*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011721:Pyrenes / Q000737:chemistry*; D051381:Rats; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D010616:Phenanthrenes; D011721:Pyrenes; C031181:phenanthrene; D003577:Cytochrome P-450 Enzyme System; C030984:pyrene; D006899:Mixed Function Oxygenases
keywords: Phanerochaete chrysosporium; biocatalyst; cytochrome P450 monooxygenase; polycyclic aromatic hydrocarbon; protein engineering
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