Evolution of the CYP2ABFGST gene cluster in rat, and a fine-scale comparison among rodent and primate species

The evolution of gene families can be best understood by studying the modern organization and functions of family members, and by comparing parallel families in different species. In this study, the CYP2ABFGST gene cluster has been characterized in rat and compared to the syntenic clusters in mouse and human, providing an interesting example of gene family evolution. In the rat, 18 loci from six subfamilies have been identified by specifically amplifying and sequencing gene fragments from cloned DNA, and have been exactly placed on chromosome 1. The overall organization of the gene cluster in rat is relatively simple, with genes from each subfamily in tandem, and is more similar to the mouse than to the human cluster. We have reconstructed the probable structure of the CYP2ABFGST cluster in the common ancestor of primates and rodents, and inferred a model of the evolution of this gene cluster in the three species. Numerous nontandem and block duplications, inversions, and translocations have occurred entirely inside the cluster, indicating that pairing between duplicate genes is keeping the rearrangements within the cluster region. The initial tandem duplication of a CYP2 gene in an early mammalian ancestor has made this region particularly subject to such localized rearrangements. Even if duplicated genes do not have a large-scale effect on chromosomal rearrangements, on a local level clustered gene families may have contributed significantly to the genomic complexity of modern mammals.     

Characterization of human CYP1A1/1A2 induction by DNA microarray and alpha-naphthoflavone

DNA microarrays and real time PCR were used to analyze the mechanism of gene induction by CYP1A1 inducers, beta-naphthoflavone, and omeprazole, in the human hepatocellular carcinoma HepG2 cells. Reproducible and significant inductions were observed in a limited number of genes including CYP1A1 and CYP1A2. Genes induced by omeprazole included several protein tyrosine kinase targets. This result confirmed that omeprazole could modulate gene expressions through protein tyrosine kinase-mediated pathway. Induction ratios were considerably different from CYP1A1 and CYP1A2 (>10-fold) to other induced genes (<5-fold). alpha-Naphthoflavone, which is known as an antagonist to 2,3,7,8-tetrachlorodibenzo-p-dioxin, inhibited the inductions of heme oxygenase 1, glutamate-cysteine ligase (modifier unit), and thioredoxin reductase by beta-naphthoflavone but not those of CYP1A1 and CYP1A2. It unexpectedly enhanced the beta-naphthoflavone-mediated CYP1A1 and CYP1A2 induction. These results suggest that the CYP1A1 and CYP1A2 genes, which share their 5(') enhancer regions, are regulated differently from the other genes.     

Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.     

Phylogenetic and functional analyses of the cytochrome P450 family 4

Cytochrome P450 family 4 (CYP4) proteins metabolize fatty acids, eicosanoids, and vitamin D and are important for chemical defense. The purpose of this study was to determine the evolutionary relationships between vertebrate CYP4 subfamilies and raise functional hypotheses regarding CYP4 subfamilies with little empirical data. 132 CYP4 sequences from 28 species were utilized for phylogenetic reconstructions by maximum likelihood and Bayesian inference. Monophyly was not found with the CYP4T and CYP4B subfamilies. CYP4V clustered with invertebrate subfamilies. Evolutionary rates of functional divergence were high in pairwise comparison with CYP4X yet, comparisons with mammalian CYP4F22 genes generally had no statistically significant divergence. Radical biochemical changes were detected in regions associated with substrate binding and the active site in comparisons among the CYP4A, CYP4X, and CYP4B subfamilies. Lastly, gene expression patterns, determined in silico with EST libraries from human, chicken, frog and fish, for CYP4V was markedly different between human and actinopterygian species. Further consideration should be given to the nomenclature of the CYP4T and CYP4B subfamily genes. Strong support was seen for the placement of CYP4A as a basal subfamily to CYP4X and CYP4Z. The B, B', J', K', K″ helices and a region at the end of C-terminus were suggested as conserved regions in CYP4 genes. The function of CYP4X was hypothesized to specialize in metabolism of long chain fatty acids. CYP4F22 genes may share a similar function to other CYP4F genes, although gene expression sites were different.     

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.     

Cytochrome P450 1 genes in early deuterostomes (tunicates and sea urchins) and vertebrates (chicken and frog): origin and diversification of the CYP1 gene family

Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.     

CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis

Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.     

The new vertebrate CYP1C family: cloning of new subfamily members and phylogenetic analysis

Two novel CYP1 genes from teleost fish constituting a new subfamily have been cloned. These paralogous sequences are designated CYP1C1 and CYP1C2. Both genes were initially obtained from untreated scup Stenotomus chrysops tissues by RT-PCR and RACE. Scup CYP1C1 and CYP1C2 code for 524 and 525 amino acids, respectively, and share 80-81% identity at the nucleotide and amino acid levels. Orthologues of CYP1C1 and CYP1C2 were identified in genome databases for other fish species, and both CYP1B1 and CYP1C1 were cloned from zebrafish (Danio rerio). Phylogenetic analysis shows that CYP1Cs and CYP1Bs constitute a sister clade to the CYP1As. Analysis of sequence domains likely to have functional significance suggests that the two CYP1Cs in scup may have catalytic functions and/or substrate specificity that differ from each other and from those of mammalian CYP1Bs or CYP1As. RT-PCR results indicate that CYP1C1 and CYP1C2 are variously expressed in several scup organs.     

Characterization of cynomolgus monkey cytochrome P450 (CYP) cDNAs: is CYP2C76 the only monkey-specific CYP gene responsible for species differences in drug metabolism?

Cynomolgus monkey CYP2C76 does not have a corresponding ortholog in humans, and it is at least partly responsible for differences in drug metabolism between monkeys and humans. To determine if CYP2C76 is the only monkey-specific CYP gene, we identified cynomolgus monkey cDNAs for CYP2A23, CYP2A24, CYP2E1, CYP2J2, CYP3A5, CYP3A8, CYP4A11, CYP4F3, CYP4F11, CYP4F12, and CYP4F45. These CYP cDNAs showed a high sequence identity (93-96%) to the homologous human CYP cDNAs. The monkey CYPs were preferentially expressed in liver among the analyzed tissues. Moreover, all five analyzed monkey CYPs (CYP2A23, CYP2A24, CYP2E1, CYP3A5, and CYP3A8) metabolized typical substrates for human CYPs in the corresponding subfamilies. These results suggest that these 11 monkey CYP cDNAs are closely related to the human CYP cDNAs and thus, unlike CYP2C76, are not apparent monkey-specific cDNAs.     

Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius

Cytochrome P450 (CYP) 147F1 from Streptomyces peucetius is a new CYP subfamily of that has been identified as ω-fatty acid hydroxylase. We describe the identification of CYP147F1 as a fatty acid hydroxylase by screening for the substrate using a substrate binding assay. Screening of substrates resulted in the identification of fatty acid groups of compounds as potential hits for CYP147F1 substrates. Fatty acids from C_10:0 to C_18:0 all showed type I shift spectra indicating their potential as substrates. Among several fatty acids tested, lauric acid, myrsitic acid, and palmitic acid were used to characterize CYP147F1. CYP147F1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida as surrogate electron transfer partners. Kinetic parameters, including the dissociation constant, K_m, NADH consumption assay, production formation rate, and coupling efficiency for CYP147F1 were also determined.     

Phylogenetic Analysis of the Cytochrome P450 3 (CYP3) Gene Family

Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand vertebrate CYP3 diversity better, we determined the killifish (Fundulus heteroclitus) CYP3A30 and CYP3A56 and the ball python (Python regius) CYP3A42 sequences. We performed phylogenetic analyses of 45 vertebrate CYP3 amino acid sequences using a Bayesian approach. Our analyses indicate that teleost, diapsid, and mammalian CYP3A genes have undergone independent diversification and that the ancestral vertebrate genome contained a single CYP3A gene. Most CYP3A diversity is the product of recent gene duplication events. There is strong support for placement of the guinea pig CYP3A genes within the rodent CYP3A diversification. The rat, mouse, and hamster CYP3A genes are mixed among several rodent CYP3A subclades, indicative of a complex history involving speciation and gene duplication. Phylogenetic analyses suggest two CYP3A gene duplication events early in rodent history, with the rat CYP3A9 and mouse Cyp3a13 clade having a sister relationship to all other rodent CYP3A genes. In primate history, the human CYP3A43 gene appears to have a sister relationship to all other known primate CYP3A genes. Other, more recent gene duplications are hypothesized to have occurred independently within the human, pig, rat, mouse, guinea pig, and fish genomes. Functional analyses suggest that gene duplication is strongly tied to acquisition of new function and that convergent evolution of CYP3A function may be frequent among independent gene copies. Current address (Rachel L. Cox): Laboratory of Aquatic Biomedicine, Marine Biology Laboratory, Woods Hole, MA 02543, USA     

Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.     

A Revised Evolutionary History of the CYP1A Subfamily: Gene Duplication, Gene Conversion, and Positive Selection

Members of cytochrome P450 subfamily 1A (CYP1As) are involved in detoxification and bioactivation of common environmental pollutants. Understanding the functional evolution of these genes is essential to predicting and interpreting species differences in sensitivity to toxicity caused by such chemicals. The CYP1A gene subfamily comprises a single ancestral representative in most fish species and two paralogs in higher vertebrates, including birds and mammals. Phylogenetic analysis of complete coding sequences suggests that mammalian and bird paralog pairs (CYP1A1/2 and CYP1A4/5, respectively) are the result of independent gene duplication events. However, comparison of vertebrate genome sequences revealed that CYP1A genes lie within an extended region of conserved fine-scale synteny, suggesting that avian and mammalian CYP1A paralogs share a common genomic history. Algorithms designed to detect recombination between nucleotide sequences indicate that gene conversion has homogenized most of the length of the chicken CYP1A genes, as well as the 5′ end of mammalian CYP1As. Together, these data indicate that avian and mammalian CYP1A paralog pairs resulted from a single gene duplication event and that extensive gene conversion is responsible for the exceptionally high degree of sequence similarity between CYP1A4 and CYP1A5. Elevated nonsynonymous/synonymous substitution ratios within a putatively unconverted stretch of ∼250 bp suggests that positive selection may have reduced the effective rate of gene conversion in this region, which contains two substrate recognition sites. This work significantly alters our understanding of functional evolution in the CYP1A subfamily, suggesting that gene conversion and positive selection have been the dominant processes of sequence evolution. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Yves Van de Peer]     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.