Cytochrome P450 family 4 in a cockroach: molecular cloning and regulation by regulation by hypertrehalosemic hormone.
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ABSTRACT:
SUBMITTER: Bradfield JY
PROVIDER: S-EPMC51700 | biostudies-other | 1991 May
REPOSITORIES: biostudies-other
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Project description:The molecular analysis of insect hormone biosynthesis has long been hampered by the minute size of the endocrine glands producing them. Expressed sequence tags from the corpora allata of the cockroach Diploptera punctata yielded a new cytochrome P450, CYP15A1. Its full-length cDNA encoded a 493-aa protein that has only 34% amino acid identity with CYP4C7, a terpenoid omega-hydroxylase previously cloned from this tissue. Heterologous expression of the cDNA in Escherichia coli produced >300 nmol of CYP15A1 per liter of culture. After purification, its catalytic activity was reconstituted by using phospholipids and house fly P450 reductase. CYP15A1 metabolizes methyl (2E,6E)-3,7,11-trimethyl-2,6-dodecatrienoate (methyl farnesoate) to methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate [juvenile hormone III, JH III] with a turnover of 3-5 nmol/min/nmol P450. The enzyme produces JH III with a ratio of approximately 98:2 in favor of the natural (10R)-epoxide enantiomer. This result is in contrast to other insect P450s, such as CYP6A1, that epoxidize methyl farnesoate with lower regio- and stereoselectivity. RT-PCR experiments show that the CYP15A1 gene is expressed selectively in the corpora allata of D. punctata, at the time of maximal JH production by the glands. We thus report the cloning and functional expression of a gene involved in an insect-specific step of juvenile hormone biosynthesis. Heterologously expressed CYP15A1 from D. punctata or its ortholog from economically important species may be useful in the design and screening of selective insect control agents.
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Project description:Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.
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Project description:Cytochrome P450 (CYP) enzymes for which there is no functional information are considered "orphan" CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to "deorphanization", that is, identifying CYP20A1 functions and its roles in health and disease.
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Project description:In a study of juvenile hormone-regulated gene expression, we isolated an anonymous cDNA representing a message that was strongly suppressed by juvenile hormone in the fat body of the cockroach Blaberus discoidalis. The protein deduced from the cDNA sequence showed compelling resemblance in sequence to the transferrins, a superfamily of internally duplicated, 80-kDa iron-binding/transport proteins characterized from several vertebrates and, to date, one insect (the tobacco hornworm, Manduca sexta). We isolated a 78-kDa protein from cockroach hemolymph, verified its congruence with the cloned cDNA, and found that it did bind iron. The cockroach protein is a member of the transferrin superfamily based on several features, including 32-46% amino acid positional identity with transferrins whose sequences are known, internal homology, positioning of cysteine residues, and iron binding. Whereas the previously characterized insect transferrin binds one atom of iron per protein molecule, B. discoidalis transferrin binds two iron atoms as do the vertebrate transferrins. The diferric property of cockroach transferrin is consistent with presence of two sets of residues positioned appropriately for iron binding. Juvenile hormone suppressed transferrin mRNA levels drastically in the adult female cockroach.
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Project description:Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450.
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Project description:A gene cluster encoding all of the enzymes for the biosynthesis of the antibiotic pentalenolactone (1) has recently been identified in Streptomyces avermitilis. The biosynthetic gene cluster contains the ptlI (SAV2999) gene which encodes a cytochrome P450 (CYP183A1). PtlI was cloned by PCR and expressed in Escherichia coli as a C-terminal His6-tag protein. Recombinant PtlI bound pentalenene (3) with high affinity (KD = 1.44 +/- 0.14 muM). Incubation of recombinant PtlI with (+/-)-3 in the presence of NADPH, E. coli flavodoxin and flavodoxin reductase, and O2 resulted in conversion to a single enantiomer of pentalen-13-al (7), by stepwise allylic oxidation via pentalen-13-ol (6). The steady-state kinetic parameters for the oxidation of pentalenene (3) to pentalen-13-ol (6) were kcat = 0.503 +/- 0.006 min-1 and Km = 3.33+/-0.62 muM for 3.
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Project description:Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera.
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Project description:The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.
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Project description:Steroid hormones modulate numerous physiological processes in various higher organisms. Research on the physiology, biosynthesis, and metabolic degradation of steroid hormones is crucial for developing drugs, agrochemicals, and anthelmintics. Most steroid hormone biosynthetic pathways, excluding those in insects, have been elucidated, and the roles of several cytochrome P450s (CYPs, P450s), heme (iron protoporphyrin IX)-containing monooxygenases, have been identified. Specifically, P450s of the animal steroid hormone biosynthetic pathways and their three dimensional structures and reaction mechanisms have been extensively studied; however, the mechanisms of several uncommon P450 reactions involved in animal steroid hormone biosynthesis and structures and reaction mechanisms of various P450s involved in plant and insect steroid hormone biosynthesis remain unclear. Recently, we determined the crystal structure of P450 responsible for the first and rate-determining step in brassinosteroids biosynthesis and clarified the regio- and stereo-selectivity in the hydroxylation reaction mechanism. In this review, we have outlined the general catalytic cycle, reaction mechanism, and structure of P450s. Additionally, we have described the recent advances in research on the reaction mechanisms of steroid hormone biosynthesis-related P450s, some of which catalyze unusual P450 reactions including C-C bond cleavage reactions by utilizing either a heme-peroxo anion species or compound I as an active oxidizing species. This review article is an extended version of the Japanese article, Structure and mechanism of cytochrome P450s involved in steroid hormone biosynthesis, published in SEIBUTSU BUTSURI Vol. 61, p. 189-191 (2021).
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