Selection and Characterization of Human Cytochrome P450 1A2 Mutants with Altered Catalytic Properties

Abstract

Random mutagenesis is an approach that has the potential to provide useful information about cytochrome P450 (P450) enzymes but has not been extensively used to date. We applied our previously developed systems for generation of random libraries of human P450 1A2 with the putative substrate recognition sequences mutated (individual residues) and an Escherichia coli genotoxity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). A total of 27 mutants were screened from 6000 clones, a small portion of the library. The sequence changes were identified, and E. coli membranes containing each P450 (with NADPH-P450 reductase expressed using a bicistronic vector) were used to determine kcat and Km values for 7-ethoxyresorufin and phenacetin O-deethylation and the (in vitro) activation of MeIQ with another bacterial genotoxicity system (Salmonella typhimurium umu). Within each assay, the values of kcat/Km varied by 2 orders of magnitude, and in some cases these parameters were 3-4-fold higher than for the native enzyme. The profiles of the mutants varied considerably for the three different reactions. Some of the mutants in the Asp-320 region may be compared with site-directed mutants of rat P450 1A2 already reported, with several differences noted. Other mutants have not been studied before in human P450 1A2 or homologues and are of interest; i.e., all Phe-226 mutants showed considerably reduced activity but Glu-225 mutants had increased catalytic activities. In principle, this approach may be applied to random mutagenesis of any enzyme that converts chemicals to mutagens and can be expressed in bacteria.

Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2

“…However, our ability to predict the cooperative behavior of other ligands is still limited. One can ask the question of why phenacetin O-deethylation is apparently not cooperative (at least for human P450 1A2) (85). Why are the homotropic cooperativity patterns of pyrene and the 1-alkoxy-4-nitrobenzenes rather opposite in their appearance (Fig.…”

Six Novel NonsynonymousCYP1A2Gene Polymorphisms: Catalytic Activities of the Naturally Occurring Variant Enzymes

“…The Guengerich research group defined six substrate recognition sequence (SRS) regions of CYP1A2 based on the amino acid sequence alignment of CYP2 family members (Gotoh, 1992) (Parikh et al, 1999). Parikh et al (1999) performed a large random mutagenesis study in the SRS regions. An acidic residue, 320D, was thought to stabilize the I-helix in the heme distal region and to be required for oxygen activation.…”

“…Shimizu et al (1994) suggested that rat 318E and 319T, present in the distal site, serve as a proton source for the heterolytic scission of hydroperoxides as in the wild-type CYP1A2. Parikh et al (1999) also discussed that 226F was a critical amino acid because the k cat /K m was extremely reduced by the F226Y substitution, despite a relatively small modification. The authors pointed out that the CYP1A2 active site-forming sequences were not limited to the six SRS regions.…”

Differences in Functional Clustering of Endogenous and Exogenous Substrates Between Members of the CYP1A Subfamily

“…Leu123 and Thr127 are located within substrate recognition site 1 (SRS 1) [39], and Leu382 is one of the 22 residues lining the active site cavity as seen in human CYP1A2 structure. Leu123 and Thr127 are close to residues that were found implicated in controlling activity levels but not the substrate specificity in previous mutagenesis experiments [40,41]. Leu123 is found contiguous with two residues, Ser122 and Thr124 that line the CYP1A2 catalytic cavity, so some steric effects could also occur.…”