Cytochrome P450 Sequences Research Articles

Hydroxycinnamoyltransferase and CYP98 in phenolic metabolism in the rosmarinic acid-producing hornwort Anthoceros agrestis

Main conclusionAnthoceros agrestis hydroxycinnamoyltransferase accepts shikimic and 3-hydroxyanthranilic acids while hydroxycinnamoylester/amide 3-hydroxylase (CYP98A147) preferred p-coumaroyl-(3-hydroxy)anthranilic acid compared to the shikimic acid derivative. Alternative pathways towards rosmarinic acid have to be considered.Rosmarinic acid (RA) is a well-known ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. In the search for enzymes involved in RA biosynthesis in the hornwort Anthoceros agrestis, the hydroxycinnamoyltransferase sequence with the highest similarity to rosmarinic acid synthase from Lamiaceae has been amplified and heterologously expressed in Escherichia coli. In parallel, the single cytochrome P450 sequence belonging to the CYP98 group in Anthoceros agrestis was isolated and expressed in Saccharomyces cerevisiae which did not result in protein formation. Codon optimization and co-expression with NADPH:cytochrome P450 reductase (CPR) from Coleus blumei resulted in the formation of active enzymes. Both, the hydroxycinnamoyltransferase and CYP98 were characterized with respect to their temperature and pH optimum as well as their substrate acceptance. The hydroxycinnamoyltransferase (AaHCT6) readily accepted p-coumaroyl- and caffeoyl-CoA with a slightly higher affinity towards p-coumaroyl-CoA. The best acceptor substrate was shikimic acid (Km 25 µM with p-coumaroyl-CoA) followed by 3-hydroxyanthranilic acid (Km 153 µM with p-coumaroyl-CoA). Another accepted substrate was 2,3-dihydroxybenzoic acid. Anthranilic acid and 4-hydroxyphenyllactic acid (as precursor for RA) were not used as substrates. p-Coumaroylesters and -amides are substrates hydroxylated by CYP98 hydroxylases. The only CYP98 sequence from Anthoceros agrestis is CYP98A147. The best substrates for the NADPH-dependent hydroxylation were p-coumaroylanthranilic and p-coumaroyl-3-hydroxyanthranilic acids while p-coumaroylshikimic and p-coumaroyl-4-hydroxyphenyllactic acids were poor substrates. The biosynthetic pathway towards rosmarinic acid thus still remains open and other enzyme classes as well as an earlier introduction of the 3-hydroxyl group to afford the caffeic acid substitution pattern must be taken into consideration.

Withania somnifera: Diversity and phylogeny - An overview

The diversity and phylogenetic relationships within Solanaceae are interesting. Withania, despite being an important genus of the family, has not found proper attention for such studies and even species delimitation within the genus is debatable. Different authors at different points of times classified Withania somnifera into different morphological forms and split it into two different species. Similarly, there are various uncertainties about phylogenetic relationships below the family level in Solanaceae and Withania . In the present study, morphological and molecular data have been used to overview the diversity and phylogeny of Withania, particularly W. somnifera . The morphological studies, RAPD, ISSR, cytochrome P450 sequence, derived primers, SSR analogues, and ITS sequence data support that the species has a lot of polymorphism bifurcated into two well-demarcated varieties. However, there is no conclusive support for dividing the species into more than two forms or splitting the species into two different species, rbc L gene sequence data supports a close relationship of the genus with Physalis and associated genera.

Cytochrome P450s in the sugarcane Saccharum spontaneum

The Cytochrome P450 gene family is one of the largest in plants. Cytochrome P450 sequences have been annotated in the Saccharum spontaneum genome and they make up 1.3% of the known alleles. 394 genes have been identified in this family after mining two releases of the S. spontaneum AP85–441 genome. The P450 sequences are most similar to those of sorghum. The 45 CYP families in sugarcane have been compared to sorghum P450s. CYP51 was the most similar sequence at 98.6%. Average percent identity to the best plant blast match was 86%. Three of the 45 CYP families show modest gene expansion. CYP71 has 15 more genes CYP76 has 12 more genes and CYP81 has 6 more genes than sorghum. The other families are identical or within 4 genes, showing a strong similarity between P450s in the two species. Alleles have been assigned for this tetraploid genome. Only 32% of the genes still have four alleles.

Cytochrome P450 diversity in the tree of life

Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Priama PCR bez pou�itia izol�cie DNA ako platforma genotypovania Hedera helix, L.

Hedera helix, L. is a specie that is used for its ornamental and medicinal properties widely. In spite of its very good biochemical characterization, the knowledge about the DNA variability is very limited and no DNA markers were used to analyse the genomic variability of the ivy populations, up to date. Here, a direct PCR approach was used to evaluate a cytochrome P450 sequences based polymorphism by PBA technique. A set of 4 samples from Slovakia, 2 samples from Czech Republic, 2 samples originated in Poland, 2 samples from Croatia, 2 samples from Scotland and samples from Germany, Austria and Spain were used in analysis. Using a set of three PBA primers and their combinations, the number of amplified fragment levels obtained by individual primer combinations was for both of them 11 and the obtained polymorphism was 91% or 100% respectivelly. The PCoA analysis was performed and the genotypes belonging to a different geographic localities were grouped together.

Transcriptome Analysis Revealed Highly Expressed Genes Encoding Secondary Metabolite Pathways and Small Cysteine-Rich Proteins in the Sclerotium of Lignosus rhinocerotis.

Lignosus rhinocerotis (Cooke) Ryvarden (tiger milk mushroom) has long been known for its nutritional and medicinal benefits among the local communities in Southeast Asia. However, the molecular and genetic basis of its medicinal and nutraceutical properties at transcriptional level have not been investigated. In this study, the transcriptome of L. rhinocerotis sclerotium, the part with medicinal value, was analyzed using high-throughput Illumina HiSeqTM platform with good sequencing quality and alignment results. A total of 3,673, 117, and 59,649 events of alternative splicing, novel transcripts, and SNP variation were found to enrich its current genome database. A large number of transcripts were expressed and involved in the processing of gene information and carbohydrate metabolism. A few highly expressed genes encoding the cysteine-rich cerato-platanin, hydrophobins, and sugar-binding lectins were identified and their possible roles in L. rhinocerotis were discussed. Genes encoding enzymes involved in the biosynthesis of glucans, six gene clusters encoding four terpene synthases and one each of non-ribosomal peptide synthetase and polyketide synthase, and 109 transcribed cytochrome P450 sequences were also identified in the transcriptome. The data from this study forms a valuable foundation for future research in the exploitation of this mushroom in pharmacological and industrial applications.

Novel Detection of Insecticide Resistance Related P450 Genes and Transcriptome Analysis of the Hemimetabolous Pest Erthesina fullo (Thunberg) (Hemiptera: Heteroptera).

Erthesina fullo (Thunberg, 1783) is an economically important heteropteran species in China. Since only three nucleotide sequences of this species (COI, 16S rRNA, and 18S rRNA) appear in the GenBank database so far, no analysis of the molecular mechanisms underlying E. fullo’s resistance to insecticide and environmental stress has been accomplished. We reported a de novo assembled and annotated transcriptome for adult E. fullo using the Illumina sequence system. A total of 53,359,458 clean reads of 4.8 billion nucleotides (nt) were assembled into 27,488 unigenes with an average length of 750 bp, of which 17,743 (64.55%) were annotated. In the present study, we identified 88 putative cytochrome P450 sequences and analyzed the evolution of cytochrome P450 superfamilies, genes of the CYP3 clan related to metabolizing xenobiotics and plant natural compounds, in E. fullo, increasing the candidate genes for the molecular mechanisms of insecticide resistance in P450. The sequenced transcriptome greatly expands the available genomic information and could allow a better understanding of the mechanisms of insecticide resistance at the systems biology level.

Computational Identification and Systematic Classification of Novel Cytochrome P450 Genes in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most economically important medicinal plants. Cytochrome P450 (CYP450) genes have been implicated in the biosynthesis of its active components. However, only a dozen full-length CYP450 genes have been described, and there is no systematic classification of CYP450 genes in S. miltiorrhiza. We obtained 77,549 unigenes from three tissue types of S. miltiorrhiza using RNA-Seq technology. Combining our data with previously identified CYP450 sequences and scanning with the CYP450 model from Pfam resulted in the identification of 116 full-length and 135 partial-length CYP450 genes. The 116 genes were classified into 9 clans and 38 families using standard criteria. The RNA-Seq results showed that 35 CYP450 genes were co-expressed with CYP76AH1, a marker gene for tanshinone biosynthesis, using r≥0.9 as a cutoff. The expression profiles for 16 of 19 randomly selected CYP450 obtained from RNA-Seq were validated by qRT-PCR. Comparing against the KEGG database, 10 CYP450 genes were found to be associated with diterpenoid biosynthesis. Considering all the evidence, 3 CYP450 genes were identified to be potentially involved in terpenoid biosynthesis. Moreover, we found that 15 CYP450 genes were possibly regulated by antisense transcripts (r≥0.9 or r≤–0.9). Lastly, a web resource (SMCYP450, http://www.herbalgenomics.org/samicyp450) was set up, which allows users to browse, search, retrieve and compare CYP450 genes and can serve as a centralized resource.

Temporal and spatial regulation of anthocyanin biosynthesis provide diverse flower colour intensities and patterning in Cymbidium orchid.

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid. Cymbidium orchid (Cymbidium hybrida) has a range of flower colours, often classified into four colour groups; pink, white, yellow and green. In this study, the biochemical and molecular basis for the different colour types was investigated, and genes involved in flavonoid/anthocyanin synthesis were identified and characterised. Pigment analysis across selected cultivars confirmed cyanidin 3-O-rutinoside and peonidin 3-O-rutinoside as the major anthocyanins detected; the flavonols quercetin and kaempferol rutinoside and robinoside were also present in petal tissue. β-carotene was the major carotenoid in the yellow cultivars, whilst pheophytins were the major chlorophyll pigments in the green cultivars. Anthocyanin pigments were important across all eight cultivars because anthocyanin accumulated in the flower labellum, even if not in the other petals/sepals. Genes encoding the flavonoid biosynthetic pathway enzymes chalcone synthase, flavonol synthase, flavonoid 3' hydroxylase (F3'H), dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) were isolated from petal tissue of a Cymbidium cultivar. Expression of these flavonoid genes was monitored across flower bud development in each cultivar, confirming that DFR and ANS were only expressed in tissues where anthocyanin accumulated. Phylogenetic analysis suggested a cytochrome P450 sequence as that of the Cymbidium F3'H, consistent with the accumulation of di-hydroxylated anthocyanins and flavonols in flower tissue. A separate polyketide synthase, identified as a bibenzyl synthase, was isolated from petal tissue but was not associated with pigment accumulation. Our analyses show the diversity in flower colour of Cymbidium orchid derives not from different individual pigments but from subtle variations in concentration and pattern of pigment accumulation.

The Pharmacogenetics of Cytochrome P450 2C19 Enzymes - Effects on Clopidogrel and Proton Pump Inhibitors

BACKGROUND: Cytochrome P450 (CYP) enzymes play important roles in human, including drug metabolism. CYP2 is the largest family of human CYP, with its sequence comprising almost one third of all CYP sequences, and responsible for the metabolism of approximately 2% of clinically administrated drugs. One of the most important enzymes in this family is the CYP2C19 enzyme. The CYP2C19 gene is polymorphic, and the variation is common especially in the Asian population.CONTENT: CYP2C19 is responsible for the metabolism of various drugs, including proton pump inhibitors (PPIs) such as omeprazole and lansoprazole, psychotropic drugs including diazepam and imipramine, anticonvulsants such as phenobarbital and mephenytoin. and the recently most studied the anti-platelet drug, clopidogrel, and many others. Drugs metabolized predominantly by this enzyme like clopidogrel and PPIs might be much affected by the genotype status of CYP2C19. Clopidogrel is a pro-drug requiring a group of enzymes to convert to its active form, particularly the CYP2C19. PPIs are metabolized to its inactive metabolites mainly by CYP2C19 in the liver. Some PPIs are inhibitor of CYP2C19 enzymes, and interaction of PPIs and clopidogrel has been widely studied.SUMMARY: The association of CYP2C19 genotypes with the plasma level of active clopidogrel and platelet reactivity in individual taking this drug is well-established. Although conflicting results still exist for the association of CYP2C19 genotypes to the clinical outcomes of clopidogrel therapy, this effect seems to be consistent in patients receiving clopidogrel for coronary stents. Due to the interaction of certain PPIs and clopidogrel, the use of PPIs other than omeprazole is recommended, especially for patients taking dual anti platelet therapy of clopidogrel and aspirin.KEYWORDS: pharmacogenetics, CYP2C19, proton pump inhibitors, clopidogrel

De Novo Transcriptome Sequencing Reveals Important Molecular Networks and Metabolic Pathways of the Plant, Chlorophytum borivilianum

Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.

Cytochrome P450 1C1 complementary DNA cloning, sequence analysis and constitutive expression induced by benzo-a-pyrene in Nile tilapia (Oreochromis niloticus)

CYP1C is the newest member of the CYP1 family of P450s; however, its physiological significance, inducers, and metabolic functions are unknown. In this study, a new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from Nile tilapia (Oreochromis niloticus) liver after intracoelomic injection with benzo-a-pyrene (BaP). The full-length cDNA was 2223 base pair (bp) long and contained an open reading frame of 1581bp encoding a protein of 526 amino acids and a stop codon. The sequence exhibited 3′ non-coding region of 642bp. The deduced amino acid sequence of O. niloticus CYP1C1 shows similarities of 86, 82.5, 79.7, 78.7, 77.8, 75.5, 69.6 and 61.3% with scup CYP1C1, killifish CYP1C1,1C2, Japanese eel CYP1C1, zebra fish CYP1C1, common carp CYP1C1, scup CYP1C2, common carp CYP1C2 and zebra fish CYP1C2, respectively. Phylogenetic tree based on the amino acids sequences clearly shows tilapia CYP1C1 and scup CYP1C1 to be more closely related to each other than to CYP1C genes from other species. Furthermore, for measuring BaP induction of CYP1C1 mRNA in different organs of tilapia (O. niloticus), β-actin gene as internal control was selected based on previous studies to assess their expression variability. Real time RCR results revealed that there was a large increase in CYP1C1 mRNA in liver (43.1), intestine (5.1) and muscle (2.4).

An Expanded, Unified Substrate Recognition Site Map for Mammalian Cytochrome P450s: Analysis of Molecular Interactions Between 15 Mammalian CYP450 Isoforms and 868 Substrates

The original map of mammalian cytochrome P450 (CYP450) residues involved in substrate recognition was prepared for the CYP2 family by Gotoh in 1992 by manual alignment of mammalian CYP450 residues with substrate recognition site (SRS) residues manually delimited from a bacterial cytochrome P450-substrate complex. Using modern structural bioinformatics tools, we have identified CYP450-ligand interactions in mammalian complexes to create a "X-ray structures" SRS map. In a parallel approach, we have built a "docking" SRS map by successful docking of 868 known substrates of 10 mammalian CYP450 isoforms and analysis of contacts made in docking solutions. We subsequently combined these maps to create a unified description of SRSs. The new map largely agrees with the original map by Gotoh with the six original SRS regions appearing in similar locations along the CYP450 sequence as in Gotoh's map. However, important differences also occur: Two new SRS regions appear before SRS1 and we have assigned them as SRS1'a and SRS1'b; SRS1 is much bigger in our map than in Gotoh's (49 aligned positions versus 28); & SRS2 and SRS3 are co-joined in our map to give a single large SRS region (60 aligned positions) we have designated as SRS(2,3), in contrast to the 9 and 10 aligned positions individually covered by SRS2 and SRS3 respectively in Gotoh's original map. These differences result in the SRS zone covering 33 % of the mammalian CYP450 sequence in our map as opposed to 16 % in Gotoh's map.

Confutation of the existence of sequence-conserved cytochrome P450 enzymes in Plasmodium falciparum

The aim of the present study was to find evidence for a homologous protein of the mammalian cytochrome P450 family member CYP2B1/B2 in Plasmodium falciparum at the nucleic acid level. Prior research had demonstrated enzyme activity in the parasite comparable to mammalian CYP1A, 2A, 2B and 2E enzymes and presence of CYP enzymes by spectrophotometric and electrophoretic analyses. In recent years, the transcriptome/proteome data of P. falciparum and other Plasmodium spp. have been published and we performed an in silico analysis to identify putative cytochrome P450 family members in the parasite. This analysis failed to identify homologs to CYP1A, 2A, 2B and 2E enzymes in Plasmodium. A prior study had also claimed the presence of a conserved CYP2B1/B2 gene in the parasite by using Northern analysis with a rat CYP2B1/B2 probe. We have repeated this analysis by cloning a rat CYP2B1/B2 cDNA and using it as a hybridization probe against total RNA extracted from P. falciparum K1 and 3D7 clones but did not obtain positive results. This is consistent with the transcriptome/proteome sequence data and suggests that the genus Plasmodium contains either only highly diverged CYP proteins which are not easily identified by their primary sequence or that they have been functionally replaced by other enzymes. It is suggested that further studies are performed that allow isolation and identification of such proteins through their functional activities.

Progress in tracing the evolutionary paths of cytochrome P450

The state of cytochrome P450 sequence accumulation in different phyla is summarized. 12,456 P450s are currently named, with about 6000 more that are known, but not yet named. As the number of genomes sequenced moves from a few dozen to an inevitable few thousand, issues of nomenclature are discussed. Orthology will be the guiding principle for naming across related genomes such as vertebrates. Even if 1000 vertebrate genomes are sequenced there will still be only 19 CYP families in vertebrates. The variable clusters of genes in families CYP2, CYP3 and CYP4 may pose challenges for naming as 1:1 orthologs do not necessarily exist. The value of synteny across genomes is emphasized as a tool for deep time evolutionary studies of P450s in animals. There is evidence that macrosynteny may be useful in tracing the origin of animal CYP clans. The concept of saturation of sequence space is described and used to estimate how complete our knowledge is of P450s in different phyla. The special niche of filamentous fungal P450s acting in secondary metabolite gene clusters is discussed. From one quarter to one third of P450s in these fungi may be dedicated to these roles.

Identification of CYP genes in Mytilus (mussel) and Crassostrea (oyster) species: First approach to the full complement of cytochrome P450 genes in bivalves

Understanding the fate and effects of organic chemicals in animals requires knowledge of cytochrome P450 (CYP) genes, which thus far are poorly known in bivalve mollusks. We searched for CYP sequences in EST databases for Mytilus and Crassostrea species, lophotrochozoan representatives of the protostomes. From ESTs averaging ca. 924 bp, we identified 58 CYP genes in Mytilus californianus and 39 CYP genes in Crassostrea gigas. The sequences fell in all known animal CYP clans, and collectively they clustered in phylogenetic analysis with vertebrate CYP families 1, 2, 3, 4, 17, 20, 26 and 27. As in deuterostomes, a majority of the sequences fell in Clan 2. The CYP sequences found thus far in bivalves suggest a diversity consistent with that found in many other animal species. The present description of mollusk genes provides the overall framework for classification of any additional bivalve sequences. The sequences identified also will be useful in obtaining full-length sequences and in designing primers for analysis of expression of mussel and oyster CYP genes, or for recombinant protein expression to identify potential substrates for the bivalve CYP proteins, and understand their roles in xenobiotic detoxification and physiology of bivalves.

Comparison of Cytochrome P450 Genes from Six Plant Genomes

Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms, CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families, of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387 P450 sequences have been identified and annotated in plant databases. As no new angiosperm CYP families have been discovered since 2004, it is now apparent that the sampling of CYP diversity is beginning to plateau. This review presents a comparison of 1,415 cytochrome P450 sequences from the six sequenced genomes of Vitis vinifera (grape), Carica papaya (papaya), Populus trichocarpa (poplar), Oryza sativa (rice), Arabidopsis thaliana (Arabidopsis or mouse ear’s cress) and Physcomitrella patens (moss). An evolutionary analysis is presented that tracks land plant P450 innovation over time from the most ancient and conserved sequences to the newest dicot-specific families. The earliest or oldest P450 families are devoted to the essential biochemistries of sterol and carotenoid synthesis. The next evolutionary radiation of P450 families appears to mediate crucial adaptations to a land environment. And, the newest CYP families appear to have driven the diversity of angiosperms in mediating the synthesis of pigments, odorants, flavors and order-/genus-specific secondary metabolites. Family-by-family comparisons allow the visualization of plant genome plasticity by whole genome duplications and massive gene family expansions via tandem duplications. Molecular evidence of human domestication is quite apparent in the repeated P450 gene duplications occurring in the grape genome.

Prediction of sites under adaptive evolution in cytochrome P450 sequences and their relationship to substrate recognition sites

We have used maximum-likelihood models of codon substitution to investigate the role of adaptive evolution in the evolution of cytochrome P450 (CYP) sequences. Evidence for the operation of adaptive evolution in the evolution of rat CYP2C, rabbit CYP2C, rat CYP2D, human CYP3A and rabbit CYP4A was observed. The absence of signal in rat CYP2B, rat CYP3A, human CYP2C and monkey CYP2C suggests that the adaptive evolution did not operate in the evolution of these cytochromes. Our results show identical adaptive evolution patterns for rabbit (lagomorpha) and rat (rodentia) CYP2C. The absence of signal for adaptive evolution in primate CYP2C suggests that the identical rat and rabbit CYP2C patterns arose in the last common ancestor of rodentia and lagomorpha. Furthermore, we have found statistically significant association of sites under adaptive evolution and Gotoh's substrate recognition sites in rat and rabbit CYP2C (5%), human CYP3A and rat CYP2D (10%). From these correlations, the given substrate-dependent nature of differences in CYP substrate-specificity profiles and differences in cytochrome active-site residues, we hypothesize that the most likely role of adaptive evolution in the evolution of cytochrome P450 substrate specificities was to fix mutations that permitted an increased number of binding modes (thereby expanding the substrate repertoire). The pattern of adaptive evolution observed in this work is consistent with results from microsomal studies in which CYP2C isoforms are responsible for most of the metabolism of foreign compounds in rat and rabbit, and CYP3A isoforms play the same role in humans.

The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions

Congenital adrenal hyperplasia (CAH) [OMIM 201910] is a group of autosomal recessive disorders, caused in 90-95% of cases by a deficiency of steroid 21-hydroxylase due to mutations in the CYP21A2 gene. The functional and structural effects of a novel rare missense mutation (E351K) in CYP21A2 found in a male patient with simple virilizing CAH were studied. The novel E351K point mutation is located in the ERR triad of the 21-hydroxylase. The ERR triad is a glutamine-arginine-arginine motif conserved in all cytochrome P450 sequences. The glutamate and first arginine residue are invariant in all P450 cytochrome enzymes, whereas the second arginine residue is present as arginine, histidine, or asparagine. Although the ERR triad is involved in some way to heme binding by the cytochrome P450 monooxygenases, the E351K mutation leads to severe but not complete loss of CYP21 enzyme activity. The functional analysis in COS-7 cells revealed a reduced conversion of 17-hydroxyprogesterone to 11-deoxycortisol of 1.1+/-0.5% (SD) and of progesterone to 11-deoxycorticosterone of 1.2+/-0.3% of wild-type activity. Analyzing the artificial mutants (E351D, E351I) of the E351 residue did not show a restoration of the in vitro 21-hydroxylase activity. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. The combination of in vitro enzyme function and computerized protein analysis of the E351 residue of the CYP21 protein provides experimental evidence for the ERR triad being a fundamental structural element of cytochrome P450 enzymes.

Identification of two Nereis virens (Annelida: Polychaeta) cytochromes P450 and induction by xenobiotics

Cytochrome P450 (CYP) enzyme catalysed metabolism of xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) are known to occur in polychaetes. Yet specific polychaete CYP enzymes have so far not been identified. Here, we report two partial CYP cDNA sequences, both of 453 bp, characterised from Nereis virens. These are the first CYP sequences reported in annelids. The deduced amino acid sequences both share highest identities to mammalian CYP4F enzymes (61% and 58%), indicating membership of the CYP4 family (accordingly, referred to as CYP4(1) and CYP4(2), respectively). The CYP4(2) gene expression was significantly higher in vehicle controls (corn oil) compared to untreated controls. Clofibrate increased the expression of the CYP4(2) genes. The induction by clofibrate and corn oil indicates regulatory similarities to vertebrate CYP4 enzymes, which are primarily involved in the metabolism of endogenous compounds such as fatty acids. Crude oil and benz(a)anthracene significantly induced CYP4(2) gene expression 2.6-fold, and because CYP enzymes often are induced by their own substrates, this induction may indicate involvement of N. virens CYP4 enzymes in the detoxification of environmental contaminants such as PAHs. The present study demonstrates that these N. virens CYP genes are transcriptionally inducible, and suggests that N. virens CYP4 enzymes may be involved in the metabolism of both exogenous and endogenous compounds.