A new cytochrome P450 from Drosophila melanogaster, CYP4G15, expressed in the nervous system

A novel cytochrome P450 was isolated from Drosophila melanogaster by PCR strategy with primers deduced from the crayfish Orconectes limosus CYP4C15 sequence, which is supposed to be involved in ecdysteroid biosynthesis. The full-length cDNA contains a 1980 bp open reading frame encoding a predicted protein of 574 amino acids and was designated CYP4G15. The corresponding gene is located at 10C1 on the X chromosome. The presence of a N-terminal segment mainly hydrophobic indicated that the corresponding enzyme is probably microsomal. In situ hybridization demonstrated predominant expression of CYP4G15 in the brain of third larval instar and Northern-blots showed no overexpression in insecticide resistant strain. This is the first indication of a specific P450 expressed in the central nervous system of Drosophila, and the putative function of the corresponding enzyme is discussed.     

Characterization and tissue distribution of a novel human cytochrome P450-CYP2U1

A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members. The gene has only five exons and the enzyme harbors two insertions in the N-terminal region. Northern blot analysis revealed high mRNA expression in human thymus, with weaker expression in heart and brain, whereas in the rat similar mRNA levels were detected in thymus and brain. Western blot analysis revealed much higher CYP2U1 protein expression in rat brain than in thymus, particularly in limbic structures and in cortex. The physiological and toxicological role of this novel P450 is still unknown, but the selective tissue distribution suggests an important endogenous function.     

Molecular cloning and nucleotide sequence of a new P450 gene, CYP319A1, from the cattle tick, Boophilus microplus.

We have isolated and sequenced a novel P450 gene (CYP319A1) from the cattle tick, Boophilus microplus. The CYP319A1 cDNA encodes a protein of 531 amino acids with an estimated molecular weight of 60.9k. It contains all highly conserved motifs characteristic of P450 enzymes. Comparison of deduced amino acid sequence with other CYP members shows that the CYP319A1 is more closely related to CYP4 family, but its overall identity to the CYP4 family is less than 40%. Therefore, it was assigned to a new P450 family by the P450 nomenclature committee. A pseudogene which shares high homology with the CYP319A1 was identified. Analysis of genomic sequence of the pseudogene indicated that the pseudogene contains two additional DNA inserts in the coding region, which disrupt the open reading frame. RT-PCR analysis showed that CYP319A1 is expressed in both susceptible and acaricide-resistant ticks.     

Cloning, sequencing, heterologous expression, and characterization of murine cytochrome P450 3a25*(Cyp3a25), a testosterone 6beta-hydroxylase

A full-length cDNA clone encoding a novel form of the cytochrome P450 3A subfamily (Cyp3a-25) has been isolated from a mouse liver cDNA library. The sequence contained 2010 base pairs and encoded a protein with 503 amino acids. The amino acid sequence shared greater identities with rat CYP3A18 (90%) and golden hamster CYP3A10 (81%) sequences than with known mouse sequences (Cyp3a-11, Cyp3a-13, Cyp3a-16, and Cyp3a-41 [68--70%]). CYP3A25 was expressed in the Escherichia coli PCWori(+) expression vector following slight modifications of the N- and C-terminals of the cDNA. The purified CYP3A25 was recognized on an immunoblot by CYP3A1 antibody and has a molecular weight of 50 kD. CYP3A25 was catalytically active in the 6 beta-hydroxylation of testosterone and the N-demethylation of benzphetamine and erythromycin. It was demonstrated by RT-PCR that the CYP3A25 mRNA is present in both fetal and adult tissues, including liver, lung, intestines, kidney, and brain. Northern blotting demonstrated that expression is greatest in the liver and small intestine.     

Molecular cloning and characterization of three novel cytochrome P450 2D isoforms, CYP2D20, CYP2D27, and CYP2D28 in the Syrian hamster (Mesocricetus auratus)

We cloned three novel cytochrome P450 (CYP) 2D cDNAs in the Syrian hamster (Mesocricetus auratus). Each clone contained an open reading frame of 1500 nucleotides encoding a protein of 500 amino acids. The deduced amino acid sequences of these had high identities with those of the other CYP2D members, therefore, the clones were assigned as CYP2D20, CYP2D27, and CYP2D28. Northern blot analysis showed that the CYP2D27 mRNA was expressed in liver, but not in kidney, small intestine, and brain, while the CYP2D20 and CYP2D28 mRNAs were not detected in these tissues examined. The expression of CYP2D27 mRNA in liver did not show sex difference and was not induced by either 3-methylcholanthrene or phenobarbital treatment. We characterized the enzyme activities of recombinant CYP2D27 expressed in COS-7 cells. The CYP2D27 protein had the bufuralol 1'-hydroxylase and debrisoquine 4-hydroxylase activities that are specific to the CYP2D subfamily.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

CYP7B1: One Cytochrome P450, Two Human Genetic Diseases, and Multiple Physiological Functions

The CYP7B1 cytochrome P450 enzyme hydroxylates carbons 6 and 7 of the B ring of oxysterols and steroids. Hydroxylation reduces the biological activity of these substrates and facilitates their conversion to end products that are readily excreted from the body. CYP7B1 is expressed in the liver, reproductive tract, and brain and performs different physiological functions in each tissue. Hepatic CYP7B1 activity is crucial for the inactivation of oxysterols and their subsequent conversion into bile salts. Loss of CYP7B1 activity is associated with liver failure in children. In the reproductive tract, the enzyme metabolizes androgens that antagonize estrogen action; mice without CYP7B1 have abnormal prostates and ovaries. The role of CYP7B1 in brain is under investigation; recent studies show that spastic paraplegia type 5, a progressive neuropathy, is caused by loss-of-function mutations in the human gene.There are two general types of cytochrome P450 enzymes, those that act on endogenous substrates like lipids and those whose primary function is to act on exogenous substrates like drugs and environmental chemicals. Deciding whether a new P450 acts on endogenous versus exogenous substrates is difficult in the absence of experimental analysis. This was the case for the P450 now known as CYP7B1, whose cDNA was initially isolated from a rat hippocampal library and described in the Journal of Biological Chemistry as a new P450 with 39% sequence identity to cholesterol 7α-hydroxylase (CYP7A1) (1). The latter enzyme acts on an endogenous substrate (cholesterol) and plays an important role in bile salt synthesis (2), but what role did CYP7B1 play? Subsequent biochemical experiments showed that CYP7B1 also acted on endogenous substrates, hydroxylating dehydroepiandrosterone, pregnenolone, and other steroids (3) as well as two oxysterols, 25-hydroxycholesterol (4) and 27-hydroxycholesterol (5). Here, we review the biochemistry, physiology, and genetics of CYP7B1, which reveal the enzyme to be a multifunctional P450.     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59 136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine Received: 18 February 1997 / Accepted: 18 May 1997     

Cloning and overexpression of CYP6F1, a cytochrome P450 gene, from deltamethrin-resistant Culex pipiens pallens

CYP6F1 (GenBank/EMBL accession No. AY662654), a novel gene with a complete encoding sequence in the cytochrome P450 family 6, was cloned and sequenced from deltamethrin-resistant 4th instar larvae of Culex pipiens pallens. The cDNA sequence of CYP6F1 has an open reading frame of 1527 bp, which encodes a putative protein of 508 amino acid residues. The deduced amino acid sequence of CYP6F1 indicated that the encoded P450 has conserved domains of a putative membrane-anchoring signal,putative reductase-binding sites, a typical heme-binding site, an ETLR motif and substrate recognition sites.Semi-quantitative RT-PCR analysis indicated that the CYP6F1 gene was expressed to a greater extent in the deltamethrin-resistant strain than in the susceptible strain of Cx. pipiens pallens. The expression levels of the CYP6F1 gene in the deltamethrin-resistant 1 st, 2nd, 3rd, 4th instar larvae and adult female mosquitoes differed, with highest expression levels in the 4th instar larvae. In addition, the CYP6F1 gene was stably expressed in mosquito C6/36 cells, and the expected 61.2 kDa band was identified by Western blotting. The cells transfected with CYP6F1 had an increased resistance to deltamethrin as compared with control cells.These results indicate that CYP6F1 is expressed at higher levels in the deltamethrin-resistant strain, and may confer some insecticide resistance in Cx. pipiens pallens.     

Structural characterization of the gene and corresponding cDNA for the cytochrome P450rm from Rhodotorula minuta which catalyzes formation of isobutene and 4-hydroxylation of benzoate

Cytochrome P450rm was previously isolated from the basidiomycete yeast Rhodotorula minuta as a bifunctional enzyme with isobutene-forming and benzoate 4-hydroxylase activities. We cloned the gene and corresponding cDNA for P450rm in order to characterize the enzyme in the context of fungal phylogeny and physiology. From the cDNA sequence, P450rm was deduced to have 527 amino acids with a calculated molecular weight of 59 136. P450rm shared 48% amino acid sequence identity with CYP53A1 from Aspergillus niger, indicating that the gene belongs to a novel subfamily of CYP53, CYP53B. However, the organization of the P450rm gene, which has eight exons and seven introns, differed completely to that of CYP53A1. Northern analysis demonstrated that the level of P450rm mRNA expression increased when L-phenylalanine was used as sole carbon source. These results suggest that P450rm has been well conserved during the evolution of fungi as a benzoate 4-hydroxylase in the dissimilation pathway starting from L-phenylalanine     

Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family,from cultured Coptis japonica cells

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Molecular cloning, heterologous expression, and characterization of a novel member of CYP2A in the Syrian hamster.

The cDNA clone coding for a novel cytochrome P-450 2A subfamily member (CYP2A16) was isolated from a Syrian hamster liver cDNA library. The deduced amino acid sequence of CYP2A16 showed more than 90% identity with those of rat CYP2A3 and mouse CYP2A4/5. The catalytic activity of CYP2A16 was determined by transient expression of its cDNA in transfected COS7 cells and CYP2A16 was found to have the testosterone 2 beta-, 15 alpha-, and 15 beta-hydroxylases, coumarin 7-hydroxylase, and ethoxycoumarin O-deethylase activities. These enzymatic characteristics of CYP2A16 are different from those of other Syrian hamster CYP2A subfamily members, CYP2A8 and CYP2A9. Northern blot analysis showed that CYP2A16 was expressed in kidney and lung while most of the other CYP2A subfamily members have been reported to be expressed in liver and olfactory. These observations indicated that the Syrian hamster CYP2A16 had unique properties compared with those of other CYP2A subfamily members.     

Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.

Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.     

CYP9E2, CYP4C21 and related pseudogenes from German cockroaches, Blattella germanica: implications for molecular evolution, expression studies and nomenclature of P450s

The cDNAs of two novel P450s (CYP9E2 and CYP4C21) were isolated from German cockroaches, Blattella germanica. Both CYP9E2 and CYP4C21 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Northern blot analyses using a CYP9E2 or CYP4C21 probe showed that 'CYP9E2' and 'CYP4C21' were expressed at all life stages. Two pseudogenes related to CYP9E2 and three pseudogenes related to CYP4C21 were also isolated. These represent the first P450 pseudogenes from an insect other than Drosophila melanogaster. The relative number of P450 pseudogenes in B. germanica is apparently higher than in D. melanogaster. The implications of these results for the molecular evolution, expression studies and nomenclature of P450s are discussed.