Abstract
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.
Footnotes
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This study was supported by Australian Research Council Discovery Project DP210635 (to E.M.J.G. and J.J.D.V.) and by Australian Research Council Linkage Project LP560595 (to E.M.J.G., J.J.D.V., and M.A.H.) with AstraZeneca R&D Mölndal, Sweden.
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This study was presented in preliminary form at the 16th International Symposium on Microsomes and Drug Oxidations, Budapest, Hungary, September 3 to 7, 2006.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017939.
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ABBREVIATIONS: P450, cytochrome P450; hNPR, human NADPH-cytochrome P450 reductase; luciferin-CEE, luciferin 6′-chloroethyl ether; luciferin-ME, luciferin 6′-methyl ether; luciferin-H, 6′-deoxyluciferin; luciferin ME-EGE, the ethylene glycol ester of luciferin 6′-methyl ether; luciferin-BE, luciferin 6′-benzyl ether; PNP, p-nitrophenol; 7-ER, 7-ethoxyresorufin; 7-PR, 7-pentoxyresorufin; PCR, polymerase chain reaction; EROD, 7-ethoxyresorufin O-deethylase; PROD, 7-pentoxyresorufin O-depentylase.
- Received July 29, 2007.
- Accepted September 5, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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