A shuffled CYP1A library shows both structural integrity and functional diversity.
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.
Johnston
Wayne A
WA
Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Australia.
Huang
Weiliang
W
De Voss
James J
JJ
Hayes
Martin A
MA
Gillam
Elizabeth M J
EM
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