The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.

Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

Sturgeon orphanin, a molecular "fossil" that bridges the gap between the opioids and orphanin FQ/nociceptin.

The elucidation of the cDNA sequence for sturgeon proorphanin provides a unique window for interpreting the evolutionary history of the opioid/orphanin gene family. The molecular "fossil" status of this precursor can be seen in several ancestral sequence characteristics that point to its origin as a duplication of either a prodynorphin- or proenkephalin-like gene. The sturgeon proorphanin cDNA encodes a precursor protein of 194 residues, and the orphanin heptadecapeptide itself binds not only the opioid receptor-like 1 (ORL1) receptor but also the classical (mu, kappa, and delta) opioid receptors with near equal affinity. Allowing for this broad receptor specificity are several amino acid substitutions at key positions in the heptadecapeptide sequence, relative to its mammalian orthologs, that have been linked by amino acid scans and site-directed mutagenic studies to the exclusion of mammalian orphanin FQ/nociceptin from classic opioid ligands (i.e. F1Y and L14W). The unique receptor binding profile of sturgeon orphanin not only provides insight into the evolutionary history of the opioid and opioid-related peptides but also provides an ideal context in which to investigate the underlying mechanisms by which novel and often divergent physiological functions arise in receptor-ligand systems.

Analyzing the radiation of the proenkephalin gene in tetrapods: cloning of a Bombina orientalis proenkephalin cDNA.

Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis proenkephalin. It was possible to align the amino acid sequences of proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina proenkephalin may be more similar to the proposed ancestral anuran proenkephalin sequence, than either X. laevis or S. multiplicatus proenkephalin.

Analysis of fragment homology among DNA sequences from cytochrome P450 families 4 and 6.

Cytochrome P450s comprise a diverse superfamily of proteins that often share as little as 12% amino acid identity. Accordingly, the identification of novel gene families, subfamilies and alleles has been based primarily on 'benchmark' levels of global amino acid identity and, more recently, phylogenetic relatedness has been used to resolve ambiguous relationships. However, PCR-based cloning strategies have resulted in a large increase in the number of short DNA sequences, particularly among insects. Many of these fragments remain unnamed and even their gene family membership remains unknown due to the uncertainty as to whether these fragments accurately reflect the levels of sequence identity or patterns of evolutionary divergence exhibited by groups of full-length P450 sequences. As a result, the nature of P450 diversity among insects remains obscure. In this paper, P450 sequences belonging to the two major gene families, that is, families CYP4 and CYP6, are analyzed by comparing segments to full-length sequences. A parameter called the Segmental Divergence Index is used to characterize segments of P450s with respect to the degree to which they mirror the divergence of full-length sequences. The reliability of these fragments in phylogenetic analyses is also tested. The results of these analyses support the use of some commonly amplified P450 gene fragments for naming new P450 genes and for studies of P450 diversity and evolution.

Duplication of the POMC gene in the paddlefish (Polyodon spathula): analysis of gamma-MSH, ACTH, and beta-endorphin regions of ray-finned fish POMC.

The proopiomelanocortin (POMC) gene, which encodes the common precursor for MSH-related and beta-endorphin-related end products, appeared early in chordate evolution and features a variety of lineage-specific modifications. Key among these has been the apparent degeneration and subsequent deletion of the gamma-MSH region during the evolution of POMC in the ray-finned fish. A second area of increasing focus has been the role of gene duplication in the evolution of POMC in particular and the opioid/orphanin gene family in general. The cloning and phylogenetic analysis of two POMC cDNAs from the paddlefish (Polyodon spathula) is reported here and biochemical data on their processed end products are presented. Based on conceptual amino acid translations, the paddlefish cDNAs encode all functional domains and, in most cases, the flanking paired-basic amino acid cleavage sites characteristic of gnathostome POMCs (i.e., signal sequence, gamma-MSH-like region, ACTH (alpha-MSH and CLIP), gamma-LPH, beta-MSH, and beta-endorphin). Phylogenetic analysis of the paddlefish POMC sequences in the context of the duplicated POMCs of sturgeon and salmonids suggests that degeneration of the gamma-MSH core sequence and its amino-terminal proteolytic cleavage site was initiated prior to divergence of the sturgeon and paddlefish lineages over 150 mya. Finally, a comparison of the relative rates of evolutionary divergence between paralogously related POMC genes within chondrostean and salmonid lineages provides potential support for the hypothesis that some taxa (e.g., the Chondrosteii) represent relic species as a result of an exceptionally slow rate of evolutionary change.

Diversity of expressed cytochrome P450 genes in the adult Mediterranean Fruit Fly, Ceratitis capitata.

The cytochrome P450s comprise a superfamily of mostly microsomal haemoproteins which play a dominant role in the metabolism of a variety of endogenous and foreign compounds. The use of a degenerate PCR primer targeted to the haem-binding decapeptide unique to the cytochrome P450 superfamily resulted in the identification of 14 novel cytochrome P450s in the Mediterranean Fruit Fly, Ceratitis capitata. Analysis of the relative frequency of individual isoforms within the pool of isolated sequences suggests that the CYP4 and CYP6 P450 families contain the most highly expressed isoforms in adult C. capitata. Phylogenetic analyses of the conceptual amino acid translations of PCR-amplified cDNAs provides evidence that one of isolated sequences may represent a new P450 family.

Molecular evolution of the opioid/orphanin gene family.

Gene duplication is a recurring theme in the evolution of vertebrate polypeptide hormones and neuropeptides. These duplication events can lead to the formation of gene families in which divergence of function is the usual outcome. In the case of the opioid/orphanin family of genes, duplication events have proceeded along two paths: (a) an apparent duplication of function as seen in the analgesic activity of Proenkephalin and Prodynorphin end-products; and (b) divergence of function as seen in the nociceptic activity of Proorphanin end-products or the melanocortin (color change and chronic stress regulation) activity of Proopiomelanocortin end-products. Although genes coding for Proopiomelanocortin, Proenkephalin, Prodynorphin, and Proorphanin have been extensively studied in mammals, the distribution and radiation of these genes in nonmammalian vertebrates is less well understood. This review will present the hypothesis that the radiation of the opioid/orphanin gene family is the result of the duplication and divergence of the Proenkephalin gene during the radiation of the chordates. To evaluate the Proenkephalin gene duplication hypothesis, a 3'RACE procedure was used to screen for the presence of Prodynorphin-related, Proenkephalin-related, and Proorphanin-related cDNAs expressed in the brains of nonmammalian vertebrates.

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila: evidence for involvement in host plant utilization.

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3' RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs.

Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome p450s: evidence for involvement in host-plant allelochemical resistance.

Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3' RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect-host-plant relationships through xenobiotic detoxification.

Isolation and sequence analysis of cytochrome P450 12B1: the first mitochondrial insect P450 with homology to 1 alpha,25 dihydroxy-D3 24-hydroxylase.

The biosynthesis of steroid hormones is an integral component of insect growth, development and reproduction. Although there is an abundance of biochemical data implicating both microsomal and mitochondrial cytochrome P450s in steroid metabolism, molecular genetic information on mitochondrial P450s is almost entirely limited to vertebrate sequences. In the current study, a degenerate polymerase chain reaction (PCR) primer was targeted to the highly conserved region of P450 genes that encodes the heme-binding decapeptide. Using a 5' rapid amplification of cDNA ends (RACE) approach, seven novel cytochrome P450 genes were isolated from Drosophila acanthoptera, including one sequence (CYP12B1) with high regional homology to vertebrate mitochondrial P450s. Sequence analysis of the conceptual translation of the full length gene, obtained by 5'RACE, revealed an amphipathic NH2-terminus rich in basic and hydrophilic amino acids, a characteristic feature of mitochondrial P450s that distinguishes them from their distantly related microsomal relatives. Phylogenetically, CYP12B1 appears to be most closely related to the mammalian mitochondrial P450s of the CYP24 family that play a critical role in calcium homeostasis through the metabolism of vitamin D3. The potential biological role of CYP12B1 is discussed in the light of what is currently known about arthropod calcium binding proteins and their regulation.

Comparison of Drosophila cytochrome P450 metabolism of natural and model substrates.

Metabolism of some insecticides and toxic natural plant compounds is known to involve cytochrome P450 enzymes. Correlations between insecticide resistance and deethylation of the model substrate, 7-ethoxycoumarin, have prompted its use in screens for potentially resistant insect populations. The applicability of this model substrate as an indicator of the enzyme activities and inductive responsiveness of cytochrome P450 isoforms involved in the metabolism of carnegine was investigated. This toxic isoquinoline alkaloid is found in the host-plants of some species of cactophilic Drosophila. The results show that the ethoxycoumarin (ECOD) assay does not accurately predict carnegine metabolism either quantitatively or with respect to the overall pattern of activity. Therefore, the ECOD assay may be as isozyme-specific in insects as has already been demonstrated in mammals and its use as an indicator of general P450 activity is questionable.

Alkaloid metabolism by cytochrome P-450 enzymes in Drosophila melanogaster.

The cytochrome P-450 family of enzymes is the primary means of foreign compound detoxification in virtually all organisms. Cytochrome P-450s have been strongly implicated in the metabolism of cactus alkaloids, and consequently, the observed patterns of host plant utilization by cactophilic species of Drosophila in the Sonoran Desert. The current study looked for evidence of alkaloid-metabolizing P-450 enzymes in a non-cactophilic species, D. melanogaster. The results of in vitro metabolism assays indicate the presence of a phenobarbital-inducible P-450 in adult D. melanogaster which is capable of metabolizing alkaloids. P-450 quantification data suggest that the enhanced level of metabolism is not the result of an overall increase in total P-450 content. Results from larval viability and adult longevity studies indicate that D. melanogaster's in vitro activity does not produce an enhanced in vivo tolerance of alkaloids.

Comparison of larval and adult P-450 activity levels for alkaloid metabolism in desertDrosophila.

The cytochrome P-450 monooxygenase system has been implicated in plant utilization by at least three species ofDrosophila (D. nigrospiracula, D. mettleri, andD. mojavensis) that are endemic to the Sonoran Desert of the southwestern United States and northwestern Mexico. Basal and induced levels of total cytochrome P-450 were determined for third-instar and decapitated 2- to 5-day post eclosion adults of the three desert species. Total P-450 levels, both basal and induced for all species assayed, were significantly higher for adults than for larvae by up to 20-fold. On a per organism basis, the levels of in vitro metabolism of the cactus alkaloid, carnegine, and patterns of response to induction by cactus tissue for adult desertDrosophila approximated those of larvae. Induction by phenobarbital, however, resulted in levels of in vitro carnegine metabolism that were up to 5.6-fold higher in adults than in larvae.