Adaptations for the Oxidation of Polycyclic Aromatic Hydrocarbons Exhibited by the Structure of Human P450 1A2

Abstract

Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Here, we present the structure of human P450 1A2 in complex with the inhibitor ␣-naphthoflavone, determined to a resolution of 1.95 Å . ␣-Naphthoflavone is bound in the active site above the distal surface of the heme prosthetic group. The structure reveals a compact, closed active site cavity that is highly adapted for the positioning and oxidation of relatively large, planar substrates. This unique topology is clearly distinct from known active site architectures of P450 family 2 and 3 enzymes and demonstrates how P450 family 1 enzymes have evolved to catalyze efficiently polycyclic aromatic hydrocarbon oxidation. This report provides the first structure of a microsomal P450 from family 1 and offers a template to study further structure-function relationships of alternative substrates and other cytochrome P450 family 1 members. Enzymes of the cytochrome P450 (CYP)5 superfamily play a significant physiologic role in the detoxication of foreign compounds and the biosynthesis of endogenous compounds, including steroid hormones, bile acids, and cholesterol. The enzymes comprising P450 families 1, 2, and 3 contribute most extensively to the biotransformation of xenobiotics to more polar metabolites that are more readily excreted. In humans and most mammals, family 1 contains three well characterized P450 monooxygenases; 1A1, 1A2, and 1B1. These enzymes are generally distinguished from P450s in other families by their capacity to oxidize a variety of polynuclear aromatic hydrocarbons (PAHs).6 Moreover, the expression levels of the three enzymes are induced by exposure to PAHs (1). The induction is mediated by a ligand-activated transcription factor, the aryl hydrocarbon receptor, which is a basic-loop-helix PAS domain protein that binds to enhancer elements flanking the CYP1A1, CYP1A2, and CYP1B1 genes and stimulates transcription.The oxidation of PAHs is generally protective. However, some P450-catalyzed reactions can transform these relatively inert compounds into genotoxic metabolites that can initiate mutagenesis and cancer. Human P450 1A2 is notable among family 1 enzymes for the capacity to N-oxidize arylamines, the major metabolic process in the bioactivation of arylamines to potent mutagenic or carcinogenic compounds (2). ␣-Naphthoflavone (ANF), a prototype flavonoid, is known to competitively inhibit P450s of family 1, albeit at different concentrations, and has been used to discriminate between P450 family 1 enzymes (3). Flavonoids have gained recent interest in vi...

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

“…structure published by Sansen et al, 24 very close to the catalytic center, only 3.8 Å apart from the cocrystallized substrate a-NF and around 4 Å from the heme. In mammalian CYP structures, known up to now, the common structural features of the heme surroundings is formed by the I-helix, the substrate recognition site 5 and the BC loop.…”

“…This variant is located on the surface of the heme domain, on the proximal side of the heme (see Figure 5). On the basis of alignment and superposition of the human CYP1A2 crystal structure, 24 with the structure of the CYP BM3 heme and FMN-binding domains, 46 S298 also seems to be located near the CYPOR interaction area. The S298R amino-acid change might influence the interaction of CYP1A2 with its redox partner, explaining the observed differences.…”

“…2) is a significantly different enzyme from WT. On the basis of the human CYP1A2 structure published by Sansen et al, 24 and the description of substrate entrance and product exit channels of CYPs by Cojocaru et al, 47 it seems likely that this residue is located in a substrate entrance/product exit channel. Finally, G299S (no.…”

“…In a previous study by Zhou et al, 7 variant I386F showed a relative low expression level of holoenzyme when expressed in bacterial cells. The I386 residue is located in the substrate recognition site 5 and based on the CYP1A2 crystal Figure 5 Localization of altered amino acids of the selected CYP1A2 polymorphic variants using the human CYP1A2 crystal structure published by Sansen, et al 24 (This figure was generated using PDB file no. 2H14).…”

“…On the basis of a crystal structure published by Sansen et al 24 of CYP1A2 wild-type (WT) (CYP1A2*1), we selected mutants that seemed to be localized in the vicinity of the active center, heme, entrance/exit channel or the NADPH cytochrome P450 oxidoreductase (CYPOR) interaction zone. As such, these polymorphic forms concern T83M (CYP1A2*9), S212C (CYP1A2*12), S298R (no allele designation), G299S (CYP1A2*13), I314V (no allele designation), I386F (CYP1A2*4), C406Y (CYP1A2*5) and R456H (CYP1A2*8).…”