title: Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide.
abstract: Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, 77555, USA. sakumar@utmb.edu
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.forename: Chong S
authors.initials: CS
authors.lastname: Chen
authors.forename: David J
authors.initials: DJ
authors.lastname: Waxman
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D003374:Coumarins / Q000378:metabolism; D003520:Cyclophosphamide / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D019020:Directed Molecular Evolution; D006900:Hydroxylation; D007069:Ifosfamide / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D018906:Antineoplastic Agents, Alkylating; D003374:Coumarins; D011994:Recombinant Proteins; C056033:7-ethoxy-4-trifluoromethylcoumarin; D003520:Cyclophosphamide; D004247:DNA; D019362:Cytochrome P-450 CYP2B1; D007069:Ifosfamide