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Drug Metabolism and Disposition Fast Forward
First published on September 6, 2007; DOI: 10.1124/dmd.107.017939

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Received for publication August 3, 2007.
Revised September 2, 2007.
Accepted for publication September 5, 2007.

A SHUFFLED CYP1A LIBRARY SHOWS BOTH STRUCTURAL INTEGRITY AND FUNCTIONAL DIVERSITY

Wayne A. Johnston 1, Weiliang Huang 1, James J. De Voss 1, Martin A. Hayes 2, Elizabeth M. J. Gillam 1*

1 University of Queensland 2 AstraZeneca

* Address correspondence to: E-mail: e.gillam{at}uq.edu.au

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 ± SD) were detected per mutant. A high level of structural integrity as well as diverse functionality was 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 (luciferin ME-EGE), 7-ethoxyresorufin and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g. clone 22; nine times the CYP1A1 specific activity towards luciferin-CEE), novel activities (e.g. clone 35; activity towards luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g. clone 9; activity towards both selective substrates luciferin-ME and luciferin-CEE as well as towards 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.


Key words: CYP cloning, CYP1A, cytochrome P450, high throughput screening




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