TY - JOUR T1 - A Shuffled CYP1A Library Shows Both Structural Integrity and Functional Diversity JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 2177 LP - 2185 DO - 10.1124/dmd.107.017939 VL - 35 IS - 12 AU - Wayne A. Johnston AU - Weiliang Huang AU - James J. De Voss AU - Martin A. Hayes AU - Elizabeth M.J. Gillam Y1 - 2007/12/01 UR - http://dmd.aspetjournals.org/content/35/12/2177.abstract N2 - The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 ± 2.3 crossovers and 1.5 ± 1.5 spontaneous mutations (mean ± S.D.) were detected per mutant. A high level of structural integrity as well as diverse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6′-chloroethyl ether (luciferin-CEE), luciferin 6′-methyl ether (luciferin-ME), 6′-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6′-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35; activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries. The American Society for Pharmacology and Experimental Therapeutics ER -