Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

昆虫细胞色素P450基因的多样性、进化及表达调控

细胞色素P450单加氧酶（cytochrome P450 monooxygenases, P450s）是由多个功能相关的亚铁血红素 硫醇盐蛋白基因组成的一个基因超家族, 在各种内源和外源物质的代谢中起着主要作用。目前GenBank中注册的昆虫P450基因序列已超过1 000个, 其中双翅目占序列总数的74%, 鳞翅目占序列总数的16%。而昆虫P450基因序列已克隆的全长序列中大部分属于CYP4和CYP6家族, 两个家族成员分别占总数的20%和45%。利用GenBank中现已注册的昆虫P450基因的cDNA全长序列进行比对并绘制进化树, 揭示不同种类昆虫P450的亲缘关系。结果显示基于P450基因的昆虫部分目的进化关系与大部分先前依据其他分子数据或形态分类学得到的昆虫系统进化关系基本吻合。现有研究表明, 细胞色素P450基因的表达可能受顺式作用元件(cis-acting element)、反式作用因子(trans-acting factor)或两者共同调控, 调控可能涉及转录增强的转录机制或mRNA稳定性增加的转录后机制。     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.     

Cytochrome P450 and actin genes expressed in Helicoverpa zea and Helicoverpa armigera: paralogy/orthology identification, gene conversion and evolution.

Molecular phylogenetic analysis was conducted using conserved cytoplasmic actin and diversified cytochrome P450 (P450) sequences isolated from Helicoverpa zea and Helicoverpa armigera, two species thought to be closely related based on allozyme analyses. These sequences were compared in turn with published sequences from other insects to gain insight into how different gene families evolve. In Bombyx mori and these Helicoverpa species, cytoplasmic actin genes are present as a pair of tandemly duplicated paralogs with coding sequence identities as high as 95.5% (B. mori), 98.9% (H. zea) and 98.5% (H. armigera) due to recent 5'-polar gene conversions. Phylogeny and interspecies comparisons assign the six actin genes into two orthologous groups: HaA3a/HzA3a/BmA3 and HaA3b/HzA3b/BmA4, which exhibit more similarities between H. zea and H. armigera than between Helicoverpa species and B. mori. Like the actin genes in H. zea, four CYP6B genes exist as two pairs of duplicated paralogs with recent 5'-polar gene conversions. Interspecific comparisons and phylogeny analysis identified three groups of orthologous CYP6B genes: H. zea CYP6B8 or CYP6B28/H. armigera CYP6B7, H. zea CYP6B27/H. armigera CYP6B6, and H. zea CYP6B9/H. armigera CYP6B2/Heliothis virescens CYP6B10. The low degree of divergence in the first two of these groups is comparable to allelic variation within a single species. These orthologous relationships and the high degrees of similarity in both actin and P450 genes strongly indicate that these Helicoverpa species are extremely closely related.     

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. Received: 14 August 1997 / Accepted: 24 March 1998     

Divergent structures of Caenorhabditis elegans cytochrome P450 genes suggest the frequent loss and gain of introns during the evolution of nematodes

The Caenorhabditis elegans genome contains more than 60 cytochrome P450 (CYP) genes. The exon-intron organizations of all of the available and potentially active C. elegans CYP genes were inferred by a newly developed program for predicting protein-coding exons based on the alignment of a genomic DNA sequence and a protein profile. From the predicted amino acid sequences, all of the C. elegans CYP genes except one were classified into three groups, which were closely related to the mammalian drug-metabolizing P450 gene families CYP2, CYP3, and CYP4. The gene structures were strikingly divergent within each group; 20, 10, and 5 unique gene organizations were identified among 40, 18, and 5 genes in the CYP2-, CYP3-, and CYP4-related groups, respectively. The degrees of divergence in gene organization were strongly correlated with those in the amino acid sequences of encoding proteins, and the minimum rate of change in an intron insertion site was estimated to be about 90 times less frequent than amino acid substitutions. Parsimonious analyses suggested that frequent loss and gain of introns has occurred during the evolution of CYP genes in each group after the divergence of nematodes, arthropods, and deuterostomia. Few, if any, incidents of intron sliding were evident, and a model that did not allow intron insertions was highly inconsistent with the observations. All of these findings are explained better by the intron-late view than by the intron-early view.     

Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed. A total of 84 CYP-related sequences were identified and could be classified into 26 families and 47 subfamilies according to standard nomenclature. Seventy eight of the eighty four genes appear to be functional and six are probable pseudogenes. The distribution of Bombyx mori P450s in the genome shows that most of them are tandem arranged on chromosomes, only 34 genes are present as singletons, with 8 clusters including 3 or more than 3 genes. Sequence alignments were used to reconstruct phylogenetic trees and to analyze the intron-exon organizations of the functional genes. The conserved intron positioning agrees perfectly with their common grouping on the tree. The presence of three extremely ancient introns which are conserved across different clans indicates that a few introns are still highly conserved after they have undergone extensive evolutionary changes of B. mori P450 duplication and divergence. Comparison of the P450s from B. mori to the P450s from Drosophila melanogaster shows that the expansion is not uniform across the gene families. Remarkably, two mitochondrial families, the B. mori CYP333 and D. melanogaster Cyp12, formed two orthologous groups in the phylogenetic tree. All CYP333s can be proposed to be related to xenobiotic metabolism in accordance with the D. melanogaster Cyp12s. The characterization and evolutionary analysis of P450s from B. mori in the current study provide useful information for understanding the characteristics and diversity of P450s from B. mori and the baseline for functional analyses of individual P450s in this model Lepidopteran insect.     

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.     

The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There are eight genes coding for mitochondrial P450s as deduced from their homology to CYP12A1 from the house fly. The genetic map of the distribution of D. melanogaster P450 genes shows (a) the absence of P450 genes on the chromosome 4 and Y, (b) more than half of the P450 genes are found on chromosome 2, and (c) the largest cluster contains nine genes. Sequence alignments were used to draw phylogenetic trees and to analyze the intron-exon organization of each functional P450 gene. Only five P450 genes are intronless. We found 57 unique intron positions, of which 23 were phase zero, 19 were phase one and 15 were phase two. There was a relatively good correlation between intron conservation and phylogenetic relationship between members of the P450 subfamilies. Although the function of many P450 proteins from vertebrates, fungi, plants and bacteria is known, only a single P450 from D. melanogaster, CYP6A2, has been functionally characterized. Gene organization appears to be a useful tool in the study of the regulation, the physiological role and the function of these P450s.     

Multiple P450alk (cytochrome P450 alkane hydroxylase) genes from the halotolerant yeast Debaryomyces hansenii

The halotolerant alkane-assimilating yeast Debaryomyces hansenii was examined for P450 alkane hydroxylase genes known to be required for alkane assimilation in Candida. Four distinct P450alk gene segments and an allelic segment were isolated using PCR based on degenerate primers derived from the CYP52 family of alkane-inducible P450 genes. A screen of a genomic library (15–20 kb inserts) constructed for this study, using a probe based on the PCR-isolated segments, yielded seven clones. This has led to the isolation and sequence of two full-length genes DH-ALK1 and DH-ALK2. These genes, each with an ORF of 1557 bp (519 aa), contained no apparent introns and showed 64% nucleotide sequence homology (61% based on the deduced amino acid sequences). The deduced proteins had predicted molecular weights of 59,254 Da (DH-ALK1) and 59,614 Da (DH-ALK2) and have been designated CYP52A12 and CYP52A13 by the P450 Nomenclature Committee. Phylogenetic analysis based on Neighbor Joining Tree showed that DH-ALK1 and DH-ALK2 constitute new genes located on two distinct branches and are most related to the gene CYP52A3 (60% deduced aa homology) and are least related to the gene CYP52C2 (41% deduced aa homology), both of C. maltosa. The isolated genes will provide tools to better understand the diversity of the P450alk family in eukaryotic microorganisms adapted to varied environmental conditions.     

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.     

家蚕P450基因CYP18A1的克隆、序列分析及转录活性

蜕皮激素对昆虫生长、发育和繁殖有重要调控作用,尤其对蜕皮和变态过程。利用GenBank上登录的蜕皮激素C₂₆羟基化酶候选基因CYP18A1的氨基酸序列对家蚕Bombyx mori全基因组数据库进行BLASTP比对,发现了家蚕直向同源基因(ortholog),其完全编码序列经RT-PCR检测和克隆、测序验证后,再以此为信息探针检索家蚕表达序列标签(expressed sequence tags,EST)数据库进行拼接延伸,获得了一条包括5′非翻译区在内的长度为1 737 bp的cDNA序列,验证结果也表明与电子克隆序列完全一致(GenBank登录号为EF421988,P450命名委员会将其命名为CYP18A1)。该基因的开放阅读框为1 623 bp,编码541个氨基酸,含有包括P450s特征结构域在内的所有昆虫P450基因的5个保守结构域,其推定的分子量为61.67 kD,等电点为 8.54。将该基因cDNA序列与家蚕基因组序列进行比对,结果表明该基因具有6个外显子,5个内含子,外显子／内含子边界符合经典的GT-AG规则。同源性分析也发现家蚕CYP18A1与其他昆虫的直向同源基因具有较高相似性。用RT-PCR方法对家蚕主要发育变态时期与组织进行检测,显示出该基因的转录表达不仅具有时空特异性,而且在表达时期上与已报道的蚕体内蜕皮激素含量变化有紧密的一致性。该研究进一步证实了CYP18A1基因与昆虫体内蜕皮激素代谢平衡相关联。     

Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

Cloning of CYP9G2 from the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae).

Cytochrome P450s constitute a superfamily of hemoproteins, important in the metabolism of endogenous and xenobiotic compounds. The full-length cDNA of a novel cytochrome P450, CYP9G2, was isolated from a cDNA library. The cDNA is 2143 bp in length and contains an open reading frame from 50 to 1615 bp, encoding a protein of 521 amino acid residues. The putative P450 protein contains a highly hydrophobic N terminus and a P450 protein signature motif, FG/S*G*R*C*G***A/G, known as the important ligand for heme binding, analysis of the NH2-terminal sequence indicated that CYP9G2 is a microsomal P450. Using polymerase chain reaction with primers specific to CYP9G2, the genomic structure of CYP9G2 was analyzed, and it was found that the gene contains seven introns and eight exons within the coding region, all the sequences of the exon-intron junctions are consistent with the AG-GT rule. Multiple alignment indicated that CYP9G2 is most similar to CYP9E2 from the Blattella germanica (42.7% identity), it is also similar to the insect P450s in family 9, including CYP9L1 from Anopheles gambiae (38.7%) and CYP9A1 from Heliothis virescens (39.5%).     

Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution

The fugu (pufferfish) genome has been sequenced, and a second genome assembly was released 17 May 2002. Exhaustive searches were made to identify all P450 genes and pseudogenes from the earlier release of 26 October 2001. P450 genes assembled as completely as possible from these data were used to do additional searches of the newer assembly and all P450 genes and pseudogenes in the available fugu sequence data have been identified, compared to human P450s, and assigned names. There are 54 P450 genes in fugu and 1 nearly intact pseudogene (CYP3A50P). CYP1A is missing much of its N-terminal half; however, 45 P450 genes are completely assembled. Eight others are lacking only one or two exons or less. CYP2X4 is known only from an EST. This may be a 55th P450 gene if it represents an accurate sequence. In addition to 2X4, there are 16 other pseudogene fragments or small pieces of P450 genes. At the P450 family level, 17 of 18 mammalian families are found in fugu. CYP39 is the only CYP family missing and it is not seen in any other fish sequence data either. The CYP2 family shows the largest degree of divergence. In the CYP2 family, only CYP2R1 and CYP2U1 are conserved as recognizable subfamilies across species. Intron-exon boundaries are largely preserved across 420 million years of evolution.