Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding.
Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O-demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K/V193M/K170Q yielded a kcat > five times faster than wild type P450 1A2 in steady-state kinetic analysis using either isolated membrane fractions or purified, reconstituted enzymes. The enhanced catalytic activity was not attributed to changes in substrate affinity. The kinetic hydrogen isotope effect of the triple mutant did not change from wild type enzyme and suggests that C-H bond cleavage is rate-limiting in both enzymes. Homology modeling, based on an X-ray structure of rabbit P450 2C5, suggests that the locations of mutated residues are not close to the substrate binding site and therefore that structural elements outside of this site play roles in changing the catalytic activity. This approach has potential value in understanding P450 1A2 and generating engineered enzymes with enhanced catalytic activity.
Kim
Donghak
D
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 683 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232-01465, USA.
Guengerich
F Peter
FP
D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D019388:Cytochrome P-450 CYP1A2 / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D000072467:Cytochrome P450 Family 2; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D006859:Hydrogen / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames; D010078:Oxazines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013255:Steroid 21-Hydroxylase / Q000737:chemistry; D013379:Substrate Specificity
D010078:Oxazines; C076580:7-methoxyresorufin; D006859:Hydrogen; D003577:Cytochrome P-450 Enzyme System; C065407:CYP2C5 protein, Oryctolagus cuniculus; D019388:Cytochrome P-450 CYP1A2; D000072467:Cytochrome P450 Family 2; D013255:Steroid 21-Hydroxylase