Structural divergence and adaptive evolution in mammalian cytochromes P450 2C

doi:10.1016/j.gene.2006.08.017

Gene

Volume 387, Issues 1–2, 31 January 2007, Pages 58-66

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Abstract

Cytochromes P450 (CYPs) comprise a superfamily of enzymes involved in various physiological functions, including the metabolism of drugs and carcinogenic compounds present in food, making them of great importance for human health. The possibility that CYPs could be broadening or changing substrate specificity in accordance to the high diversity of xenobiotics compounds environmentally available suggests that their metabolic function could be under adaptive evolution. We evaluated the existence of functional divergence and signatures of selection on mammalian genes from the drug-metabolizing CYP2 family. Thirteen of the sites found to be functionally divergent and the eight found to be under strong positive selection occurred in important functional domains, namely on the substrate entrance channel and within the active site. Our results provide insight into CYPs evolution and the role of molecular adaptation in enzyme substrate-specificity diversification.

Introduction

Cytochromes P450 (CYPs) can be found in almost all organisms and present a high intra and interspecies diversity (Werck-Reichhart and Feyereisen, 2000). One of their tasks is the oxidation of xenobiotic compounds to facilitate their excretion from the organism (Omiecinski et al., 1999, Guengerich, 2001). These include drugs and carcinogenic compounds present in food (Anzenbacher and Anzenbacherova, 2001), which explains their importance for pharmaceutical research.

The possibility that the CYPs performing such tasks could be broadening or changing substrate specificity in accordance to environmental changes suggests that their metabolic function could be under adaptive evolution. In fact, mosquito CYPs seem to be involved in insecticide resistance (Ranson et al., 2002). Different evolutionary pressures on particular sites of CYPs should be revealed by the analysis of the rate of substitutions occurring within coding regions.

Protein functional divergence after gene duplication can be tested by calculating the replacement rates that occur in the originated subfamilies (Gaucher et al., 2001). Likelihood-ratio tests (LRTs) can be used to assess whether the rates differ between the subfamilies and/or are accelerated in one/all of them (Knudsen and Miyamoto, 2001, Gu, 2003, Knudsen et al., 2003).

Nonsynonymous substitutions may influence the fitness of an individual or population. Thus, adaptive molecular evolution may cause the nonsynonymous substitution rate (d_N) to be higher than the synonymous rate (d_S), with the ratio ω (d_N/d_S) being higher than 1 (Yang et al., 2000). The methods that provide statistical measures of such mutation rates fail, however, in evaluating the physicochemical importance of the correspondent amino acid changes and its consequences for the protein function (McClellan et al., 2005). Further analyses are necessary to determine if there is a statistically significant change in the amino acid properties at particular sites. CYPs chemical and structural variations in areas that are in contact with the ligands (substrate recognition sites, SRSs (Gotoh, 1992)) will have consequences on the size, shape, and chemical characteristics of both substrates and products.

In this study, we assessed functional divergence among mammalian CYP2C and used two types of methods to determine if these genes are under adaptive evolution: (i) a gene level approach, testing for functional divergence and positive selection using statistical methods and (ii) a protein level approach, evaluating statistically significant physicochemical amino acid changes and verifying the impact of the previous analyses on the protein three-dimensional (3D) structure. So far, positive selection has been mostly associated with protein recognition domains, such as those involved in immune response and reproduction (for a list of examples see Table S1 in supporting information; henceforth all supporting information will be indicated by an S preceding the correspondent numbering). We retrieved unequivocal evidence that diversifying selection is acting on CYP's active site, thus providing insight to understand rapid enzyme function diversification.

Section snippets

Sequences and structures

The CYP superfamily is classified into families and subfamilies (> 40% or 55% amino acid sequence identity, respectively) (Nelson et al., 2004). The enzymes used in this study belong to the largest and most diverse of CYP families, CYP2 (Omiecinski et al., 1999). Its members are responsible for the metabolism of a variety of different pharmaceutical agents and represent more than 20% of the human liver total CYPs content (Omiecinski et al., 1999). Mutations occurring both within and outside the

Selection analyses

The first evidence of selection in CYPs was retrieved by the SWAAP sliding window approach (Fig. 1A). Very low values of d_N/d_S were observed for the heme binding areas. This is consistent with the fact that these areas are associated with the catalytic oxidative mechanism, which is common to all CYPs (Poulos, 1995). By contrast, the substrate binding areas of the active site presented some of the higher values of d_N/d_S. These include SRS-1, SRS-3, SRS-2, SRS-5 and SRS-6 (Fig. 1B).

We used

Acknowledgments

We thank the FCT (Fundação para a Ciência e Tecnologia) for a doctoral scholarship (SFRH/BD/7089/2001) for R.F. and the NFCR (National Foundation for Cancer Research) Centre for Drug Discovery, University of Oxford, U.K., for financial support.

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Structural divergence and adaptive evolution in mammalian cytochromes P450 2C

Abstract

Introduction

Section snippets

Sequences and structures

Selection analyses

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

Mol. Genet. Metab.

Genetics

Arch. Biochem. Biophys.

Curr. Opin. Struct. Biol.

Arch. Biochem. Biophys.

J. Inorg. Biochem.

Cytochromes P450 and metabolism of xenobiotics

Cell. Mol. Life Sci.

Cytochrome P450-mediated oxidation of glucuronide derivatives: example of estradiol-17 beta-glucuronide oxidation to 2-hydroxyestradiol-17 beta-glucuronide by CYP2C8

Drug Metab. Dispos.

SWAPSC: sliding window analysis procedure to detect selective constraints

Bioinformatics

The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection

Mol. Biol. Evol.

A sliding window-based method to detect selective constraints in protein-coding genes and its application to RNA viruses

J. Mol. Evol.

SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny

Comput. Appl. Biosci.

Function-structure analysis of proteins using covarion-based evolutionary approaches: elongation factors

PNAS

Functional divergence in protein (family) sequence evolution

Genetica

Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity

Chem. Res. Toxicol.

The rapid generation of mutation data matrices from protein sequences

Comput. Appl. Biosci.

A likelihood ratio test for evolutionary rate shifts and functional divergence among proteins

Proc. Natl. Acad. Sci. U. S. A.

Identification of rabbit cytochromes P450 2C1 and 2C2 as arachidonic acid epoxygenases

Molecular

Unbiased estimation of the rates of synonymous and nonsynonymous substitution

J. Mol. Evol.

Physicochemical evolution and molecular adaptation of the cetacean and artiodactyl cytochrome b proteins

Mol. Biol. Evol.

Cytochrome P4502C9: an enzyme of major importance in human drug metabolism

Br. J. Clin. Pharmacol.