title: Use of directed evolution of mammalian cytochromes P450 for investigating the molecular basis of enzyme function and generating novel biocatalysts.
abstract: Directed evolution has been successfully applied to the design of industrial biocatalysts for enhanced catalytic efficiency and stability, and for examining the molecular basis of enzyme function. Xenobiotic-metabolizing mammalian cytochromes P450 with their catalytic versatility and broad substrate specificity offer the possibility of widespread applications in industrial synthesis, medicine, and bioremediation. However, the requirement for NADPH-cytochrome P450 reductase, often cytochrome b5, and an expensive cofactor, NADPH, complicates the design of mammalian P450 enzymes as biocatalysts. Recently, Guengerich and colleagues have successfully performed directed evolution of P450s 1A2 and 2A6 initially by using colony-based colorimetric and genotoxicity screening assays, respectively, followed by in vitro fluorescence-based activity screening assays. More recently, our laboratory has developed a fluorescence-based in vitro activity screening assay system for enhanced catalytic activity of P450s 2B1 and 3A4. The studies indicate an important role of amino acid residues outside of the active site, which would be difficult to target by other methods. The approach can now be expanded to design these as well as new P450s using more targeted substrates of environmental, industrial, and medical importance.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-1031, USA. sakumar@utmb.edu
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D019020:Directed Molecular Evolution; D006801:Humans
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System