Structural comparison of cytochromes P450 2A6, 2A13, and 2E1 with pilocarpine

Abstract

Summary Human xenobiotic-metabolizing cytochrome P450 (P450) enzymes can each bind and monooxygenate a diverse set of substrates, including drugs, often producing a variety of metabolites. Additionally a single ligand can interact with multiple cytochrome P450 enzymes, but often the protein structural similarities and differences that mediate such overlapping selectivity are not well understood. Even though the P450 superfamily has a highly canonical global protein fold, there are large variations in the active site size, topology, and conformational flexibility. We have determined how a related set of three human cytochrome P450 enzymes bind and interact with a common inhibitor, the muscarinic receptor agonist drug pilocarpine. Pilocarpine binds and inhibits the hepatic CYP2A6 and respiratory CYP2A13 enzymes much more efficiently than the hepatic CYP2E1 enzyme. To elucidate key amino acids involved in pilocarpine binding, crystal structures of CYP2A6 (2.4 Å), CYP2A13 (3.0 Å), CYP2E1 (2.35 Å), and a CYP2A6 mutant enzyme, CYP2A6 I208S/I300F/G301A/S369G (2.1 Å), have been determined with pilocarpine in the active site. In all four structures, pilocarpine coordinates to the heme iron, but comparisons reveal how individual amino acids lining the active sites of these three distinct human enzymes interact differently with the inhibitor pilocarpine. Hyperlinking to databases The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) with the following codes: CYP2A6 with pilocarpine (3T3R), CYP2A6 I208S/I300F/G301A/S369G with pilocarpine (3T3Q), CYP2A13 with pilocarpine (3T3S), and CYP2E1 with pilocarpine (3T3Z).

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

“…The sp 3 hybridization of C2Ј and the (S) stereochemistry of the substrate then dictate different planes for the pyridine rings and differential interactions with Asn-297. Because Asn-297 is a key orienting feature for the CYP2A active site with several other ligands (6,(12)(13)(14)(15), it is notable that in CYP2A6 the Asn-297 side chain is rotated slightly farther away from nicotine compared with the CYP2A13 structure. Differences in the details of nicotine orientation are likely also mediated by several other substantial differences between the two active sites involving both conserved and nonconserved residues.…”

“…Protein Expression, Purification, and Crystallization-Truncated, His-tagged human CYP2A6 and CYP2A13 were expressed recombinantly in Escherichia coli, purified as described (14,15,19), and crystallized using hanging drop vapor diffusion.…”

“…Structures are available for CYP2A6 with the largely in vitro substrate coumarin and inhibitors (12)(13)(14) and of CYP2A13 bound to the in vitro substrate indole or inhibitor pilocarpine (14,15). However, no structures are available for substrates with significant pharmacological relevance, much less the key addictive and procarcinogenic agents in tobacco.…”

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

“…A minor shift in the position of Phe-298 on helix I allows the carboxylate moieties to reside in an adjacent cavity between helices I, F, and G and increases the volume of the active site from ϳ190 Å 3 for indazole to 420 -490 Å 3 for the C 8 -C 12 compounds (60). Structures of 2E1, 2A6, and 2A13 co-crystallized with pilocarpine reveal conformational changes and differences in binding interactions (61).…”

Inhibition of Carcinogen-Activating Cytochrome P450 Enzymes by Xenobiotic Chemicals in Relation to Antimutagenicity and Anticarcinogenicity

“…Since molecular docking analysis has been shown to be a useful tool for the studies of the interactions of various ligands with active sites of enzymes, such as P450s, we examined and compared the ligand-interaction energies (U values) with these 24 chemicals using reported crystal structures of CYP2A13 (4EJH), 2A13 (2P85), 2A13 (3T3S), and 2A13 (4EJG) (142)(143)(144) bound to NNK, indole, pilocarpine, and nicotine, respectively, and CYP2A6 (1Z10), 2A6 (3T3R), and 2A6 4EJJ) (145,146) bound to coumarin, pilocarpine, and nicotine, respectively (30). We first determined the U values of interaction of 2-ethynylnaphthalene, 2-ethynylphenanthrene, and 4-biphenylpropagyl ether with CYP2A13 (4EJH), CYP2A13 (2P85), CYP2A13 (3T3S), and CYP2A13 (4EJG) (Fig.…”