Activation of MelQ (2-amino-3,4-dimethylimidazo-[4,5-f]quinoline) by sequence variants of recombinant human cytochrome P450 1A2

Understanding the relationships between the sequences and catalytic activit ies of P450 enzymes that catalyze the bioactivation of mutagens and carcino gens is an important goal in mutation research, Escherichia coil strain DJ4 309 expresses recombinant human P450 1A2 and activates promutagens such of MeIQ (2-amino-3,4-dimeihylimidnro[4,5-f]quinoline), as measured by inductio n of reverse mutations detected as lacZ(+) colonies on minimal lactose (ML) plates. Pools of P450 1A2 mutants were constructed by polymerose chain rea ction (PCR) mutagenesis of putative substrate recognition sites (SRSs). Cul tures of individual clones were patched onto MeIQ/MI plates and the growth of revertant microcolonies within each patch was inspected after 2 days of incubation. Beginning with a pool of several thousand clones, we identified 25 distinct P450 1A2 SRS variants with altered activities. In this study, the MeIQ dose-responses of all the variants are reported. The implications of the results are considered with reference to published models of the pro tein's structure. (C) 2000 Wiley-Liss, Inc.