Selection of human cytochrome P450 1A2 mutants with enhanced catalytic activity for heterocyclic amine N-hydroxylation.
Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens. Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products. Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation. Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with >10-fold increased MeIQ activation based on the E. coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k(cat)/K(m)) in steady-state N-hydroxylation assays done with isolated membrane fractions. SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines. The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H(2)O(2) formation. Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate. In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein.
Kim
Donghak
D
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Guengerich
F Peter
FP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001711:Biotransformation; D002384:Catalysis; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry* / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D000072467:Cytochrome P450 Family 2; D019020:Directed Molecular Evolution; D015723:Gene Library; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006900:Hydroxylation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000737:chemistry*; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D011804:Quinolines / Q000737:chemistry*; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013255:Steroid 21-Hydroxylase / Q000737:chemistry
D006571:Heterocyclic Compounds; D009153:Mutagens; D011804:Quinolines; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; C065407:CYP2C5 protein, Oryctolagus cuniculus; D019388:Cytochrome P-450 CYP1A2; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase; C036989:2-amino-3,4-dimethylimidazo(4,5-f)quinoline