Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102
Introduction
The CYP1 family is of importance in the Phase l metabolism of many xenobiotic compounds generally characterized by structural type, namely, polycyclic aromatic hydrocarbons (PAHs), nitrogenous heterocyclics and their derivatives (Butler et al., 1989, McManus et al., 1990, Harvey, 1991, Wakabayashi et al., 1992), including (hetero)aromatic amines and amides such as caffeine and 2-acetylaminofluorene (AAF). The majority of CYP1 substrates exhibit molecular planarity due to the presence of two or more fused aromatic/heterocyclic rings and, as is clear from the above list, nitrogen frequently occurs in CYP1 substrates as part of the ring system and/or in a substituent group. The role of CYP1 enzymes in the metabolic activation of many mutagens and carcinogens, such as benzo(a)pyrene and β naphthylamine, is well established (Gonzalez et al., 1991, Munro et al., 1993, Gonzalez and Gelboin, 1994, Kawajiri and Hayashi, 1996) although CYP1-mediated oxidation does not necessarily result in the formation of carcinogenic species, and many examples of detoxifying metabolism mediated by CYP1 enzymes are known (Beresford, 1993, Rendic and Di Carlo, 1997).
In a previous study (Lewis and Lake, 1996) homology modelling of rat CYP1A1, rat CYP1A2 and human CYP1A2 was employed in order to rationalize CYP1A-mediated oxidations of a number of known substrates, and to explain selectivity differences between CYP1A1 and CYP1A2 in terms of the disposition of key amino acids within the putative active sites, and it is known that species differences exist in the activation of heterocyclic amines, for example, by CYP1A2 in rat and man (Turesky et al., 1998), whereas species variations are also apparent in CYP1A1 (Boyd et al., 1995). As far as human CYP1 enzymes are concerned, three members are known (CYP1A1, CYP1A2 and CYP1B1) with CYP1A2 representing the major form. Although varying between individuals, CYP1A2 represents, on average, around 13% of the P450 complement in human liver and, moreover, is thought to be involved in about 8% of Phase 1 drug oxidations (Rendic and Di Carlo, 1997). A gender-based variability in CYP1A2 levels has been implicated as having an association with sex differences in bladder cancer incidence (Horn et al., 1995) and it is also known that CYP1A2-null mutant mice exhibit deficient drug metabolism (Liang et al., 1996).
CYP1A1 is readily inducible by polyaromatic hydrocarbons (PAHs) in experimental rodent species [reviewed by Kawajiri and Hayashi (1996)] where extent of PAH metabolism is linked with the level of CYP1A1 induction. In contrast, CYP1A1 is poorly expressed in human liver (McManus et al., 1990, Edwards et al., 1998), being present at <1% of the total hepatic P450 complement, despite being associated with about 2.5% of drug oxidations (Rendic and Di Carlo, 1997). In Homo sapiens, CYP1A1 is essentially an extrahepatic enzyme and, in particular, is induced in the lung by tobacco smoking, where its induction has been associated with the development of pulmonary cancers (Kawajiri and Hayashi, 1996). A third human CYP1 enzyme, namely CYP1B1, has been identified which appears to belong to a separate subfamily and other CYP1B1 proteins have also been characterized in both rat and mouse (Sutter et al., 1994, Tang et al., 1996). Although CYP1B1 has only been found at relatively low levels (<1%) in several tissues (Hakkola et al., 1997), it may have an important physiological role in the metabolism of oestradiol as 4-hydroxylation is a characteristic of CYP1B1-mediated metabolism of this endogenous oestrogen (Rendic and Di Carlo, 1997), whereas CYP1B1 is also known to activate several structurally-diverse procarcinogens (Shimada et al., 1996). Table 1 summarizes the typical substrates, inhibitors and inducers of CYP1 family enzymes including one of the piscine CYP1A enzymes, CYP1A6, which has been isolated in the plaice (Leaver et al., 1993).
Phylogenetic analysis of CYP1A gene sequences (Morrison et al., 1995) indicates that there are three distinct monophyletic branches corresponding to the two mammalian enzymes, CYP1A1 and CYP1A2, with the third representing the fish CYP1A genes. It has been estimated that gene duplication of CYP1A leading to the divergence and branching of CYP1A1 and CYP1A2 may have occurred about 120 million years ago (Kawajiri and Hayashi, 1996) whereas the piscine CYP1A branch is thought to have diverged substantially earlier (Morrison et al., 1995) with CYP1B separating from the CYP1A subfamily ≈300 million years ago (Tang et al., 1996). In order to understand more completely the possible amino acid residue differences within these CYP1 sequences which may determine their substrate selectivity, we have constructed three-dimensional molecular models of four of these enzymes from rat (CYP1A1), human (CYP1B1 and CYP1A2) and plaice (CYP1A6), and have investigated their likely active site interactions with known substrates and inhibitors in each case. In this way, we hope to demonstrate that the structural requirements for CYP1-selective compounds result from specific contacts with key amino acid residues, which both bind and orientate substrates for metabolism in certain positions.
Section snippets
Methods
All CYP1 models were generated from the substrate-bound CYP102 crystal structure (Li and Poulos, 1997) following the construction of a multiple sequence alignment of several proteins in the CYP1 family against that of the CYP102 haemoprotein domain, as shown in Fig. 1. To produce this alignment, we have used the GCG package (Genetic Computer Group, Madison, Wisconsin) and, in particular, the PILEUP program for initially aligning the relevant sequences via their reported SwissProt accession
Results and discussion
Energies of the geometry-optimized structures for CYP1A1, CYP1A2, CYP1B1 and CYP1A6 were −1293, −1197, −1047 and −1279 kcal mol−1, respectively, from which it can be appreciated that stable geometries of low minimum energies have resulted in each case, and are indicative of protein tertiary structures comprising conformationally-allowed backbone and side-chain geometries. The lack of any disallowed regions in any of the minimized protein structures showed that loop insertions and amino acid
Acknowledgements
The financial support of GlaxoWellcome Research and Development Limited, Merck, Sharp and Dohme Limited, the European Union Biomed 2 programme, and the University of Surrey Foundation Fund is gratefully acknowledged by D.F.V.L. The authors would like to thank Anne Hersey (GlaxoWellcome) for supplying physicochemical data on several compounds, together with Costas Ioannides (University of Surrey) and Danny Burke (De Montfort University) for some useful discussions.
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