title: A shuffled CYP1A library shows both structural integrity and functional diversity.
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.
authors.affiliation: Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Australia.
authors.forename: Wayne A
authors.initials: WA
authors.lastname: Johnston
authors.forename: Weiliang
authors.initials: W
authors.lastname: Huang
authors.forename: James J
authors.initials: JJ
authors.lastname: De Voss
authors.forename: Martin A
authors.initials: MA
authors.lastname: Hayes
authors.forename: Elizabeth M J
authors.initials: EM
authors.lastname: Gillam
mesh_terms: D002248:Carbon Monoxide; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics* / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D032621:DNA Shuffling / Q000379:methods*; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000378:metabolism; D015723:Gene Library; D005838:Genotype; D006801:Humans; D049408:Luminescent Agents / Q000378:metabolism; D009154:Mutation; D009596:Nitrophenols / Q000378:metabolism; D010078:Oxazines / Q000378:metabolism; D010641:Phenotype; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity