Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment.

Arthropod CYPomes illustrate the tempo and mode in P450 evolution.

The great diversity of P450 genes in a variety of organisms is well documented but not well explained. The number of CYP genes in each species is highly variable and this is shown here for arthropod, mainly insect CYPomes. Pairs of recognizable orthologs are but a small portion of the CYPome, but species- or lineage-specific expansions of CYP subfamilies are consistently observed. These "blooms" of CYP genes have their origin in multiple gene duplications, although some subfamilies expand and others do not. Stochastic birth and death models of CYP gene proliferation are sufficient to explain blooms, and speciation events may play important roles in CYPome diversity between lineages. Mitochondrial clan P450s are a monophyletic group of genes that has seen several blooms in insects, but apparently not in vertebrates.

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements.

The recent assembly of the silkworm Bombyx mori genome with 432 Mb on 28 holocentric chromosomes has become a reference in the genomic analysis of the very diverse Order of Lepidoptera. We sequenced BACs from two major pests, the noctuid moths Helicoverpa armigera and Spodoptera frugiperda, corresponding to 15 regions distributed on 11 B. mori chromosomes, each BAC/region being anchored by known orthologous gene(s) to analyze syntenic relationships and genome rearrangements among the three species. Nearly 300 genes and numerous transposable elements were identified, with long interspersed nuclear elements and terminal inverted repeats the most abundant transposable element classes. There was a high degree of synteny conservation between B. mori and the two noctuid species. Conserved syntenic blocks of identified genes were very small, however, approximately 1.3 genes per block between B. mori and the two noctuid species and 2.0 genes per block between S. frugiperda and H. armigera. This corresponds to approximately two chromosome breaks per Mb DNA per My. This is a much higher evolution rate than among species of the Drosophila genus and may be related to the holocentric nature of the lepidopteran genomes. We report a large cluster of eight members of the aminopeptidase N gene family that we estimate to have been present since the Jurassic. In contrast, several clusters of cytochrome P450 genes showed multiple lineage-specific duplication events, in particular in the lepidopteran CYP9A subfamily. Our study highlights the value of the silkworm genome as a reference in lepidopteran comparative genomics.

Evolution of insect P450.

The first fully sequenced insect genomes were those of the fruitfly and the mosquito, both from the order Diptera. Now, with an increasing number and diversity of insect genomes becoming available, the diversity of insect P450 genes can be better appreciated and tentative ideas about the evolution of the CYP (cytochrome P450) superfamily in insects can be proposed. There are four large clades of insect P450 genes that existed before the divergence of the class Insecta and that are also represented by CYP families in vertebrates: the CYP2 clade, the CYP3 clade, the CYP4 clade and the mitochondrial P450 clade. P450s with known or suspected physiological functions are present in each of these clades and only a dozen genes appear to have orthologues or very close paralogues in each insect genome. P450 enzymes from each of these clades have been linked to insecticide resistance or to the metabolism of natural products and xenobiotics. In particular, insects appear to maintain a repertoire of mitochondrial P450 paralogues devoted to the response to environmental challenges.

A deficit of detoxification enzymes: pesticide sensitivity and environmental response in the honeybee.

The honeybee genome has substantially fewer protein coding genes ( approximately 11 000 genes) than Drosophila melanogaster ( approximately 13 500) and Anopheles gambiae ( approximately 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.

Gene expression profiling of Spodoptera frugiperda hemocytes and fat body using cDNA microarray reveals polydnavirus-associated variations in lepidopteran host genes transcript levels.

BACKGROUND: Genomic approaches provide unique opportunities to study interactions of insects with their pathogens. We developed a cDNA microarray to analyze the gene transcription profile of the lepidopteran pest Spodoptera frugiperda in response to injection of the polydnavirus HdIV associated with the ichneumonid wasp Hyposoter didymator. Polydnaviruses are associated with parasitic ichneumonoid wasps and are required for their development within the lepidopteran host, in which they act as potent immunosuppressive pathogens. In this study, we analyzed transcriptional variations in the two main effectors of the insect immune response, the hemocytes and the fat body, after injection of filter-purified HdIV. RESULTS: Results show that 24 hours post-injection, about 4% of the 1750 arrayed host genes display changes in their transcript levels with a large proportion (76%) showing a decrease. As a comparison, in S. frugiperda fat body, after injection of the pathogenic JcDNV densovirus, 8 genes display significant changes in their transcript level. They differ from the 7 affected by HdIV and, as opposed to HdIV injection, are all up-regulated. Interestingly, several of the genes that are modulated by HdIV injection have been shown to be involved in lepidopteran innate immunity. Levels of transcripts related to calreticulin, prophenoloxidase-activating enzyme, immulectin-2 and a novel lepidopteran scavenger receptor are decreased in hemocytes of HdIV-injected caterpillars. This was confirmed by quantitative RT-PCR analysis but not observed after injection of heat-inactivated HdIV. Conversely, an increased level of transcripts was found for a galactose-binding lectin and, surprisingly, for the prophenoloxidase subunits. The results obtained suggest that HdIV injection affects transcript levels of genes encoding different components of the host immune response (non-self recognition, humoral and cellular responses). CONCLUSION: This analysis of the host-polydnavirus interactions by a microarray approach indicates that the presence of HdIV induces, directly or indirectly, variations in transcript levels of specific host genes, changes that could be responsible in part for the alterations observed in the parasitized host physiology. Development of such global approaches will allow a better understanding of the strategies employed by parasites to manipulate their host physiology, and will permit the identification of potential targets of the immunosuppressive polydnaviruses.

Comparative genomics of insect juvenile hormone biosynthesis.

The biosynthesis of insect juvenile hormone (JH) and its neuroendocrine control are attractive targets for chemical control of insect pests and vectors of disease. To facilitate the molecular study of JH biosynthesis, we analyzed ESTs from the glands producing JH, the corpora allata (CA) in the cockroach Diploptera punctata, an insect long used as a physiological model species and compared them with ESTs from the CA of the mosquitoes Aedes aegypti and Anopheles albimanus. The predicted genes were analyzed according to their probable functions with the Gene Ontology classification, and compared to Drosophila and Anopheles gambiae genes. A large number of reciprocal matches in the cDNA libraries of cockroach and mosquito CA were found. These matches defined known and suspected enzymes of the JH biosynthetic pathway, but also several proteins associated with signal transduction that might play a role in the modulation of JH synthesis by neuropeptides. The identification in both cockroach and mosquito CA of homologs of the small ligand binding proteins from insects, Takeout/JH binding protein and retinol-binding protein highlights a hitherto unsuspected complexity of metabolite trafficking, perhaps JH precursor trafficking, in these endocrine glands. Furthermore, many reciprocal matches for genes of unknown function may provide a fertile ground for an in-depth study of allatal-specific cell physiology. ESTs are deposited in GenBank under the accession numbers DV 017592-DV 018447 (Diploptera punctata); DR 746432-DV 747949 (Aedes aegypti); and DR 747950-DR 748310 (Anopheles albimanus).

CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata.

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.

A single p450 allele associated with insecticide resistance in Drosophila.

Insecticide resistance is one of the most widespread genetic changes caused by human activity, but we still understand little about the origins and spread of resistant alleles in global populations of insects. Here, via microarray analysis of all P450s in Drosophila melanogaster, we show that DDT-R, a gene conferring resistance to DDT, is associated with overtranscription of a single cytochrome P450 gene, Cyp6g1. Transgenic analysis of Cyp6g1 shows that overtranscription of this gene alone is both necessary and sufficient for resistance. Resistance and up-regulation in Drosophila populations are associated with a single Cyp6g1 allele that has spread globally. This allele is characterized by the insertion of an Accord transposable element into the 5' end of the Cyp6g1 gene.

The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis.

The first committed step in the biosynthesis of indole glucosinolates is the conversion of indole-3-acetaldoxime into an indole-3-S-alkyl-thiohydroximate. The initial step in this conversion is catalyzed by CYP83B1 in Arabidopsis (S. Bak, F.E. Tax, K.A. Feldmann, D.A. Galbraith, R. Feyereisen [2001] Plant Cell 13: 101-111). The knockout mutant of the CYP83B1 gene (rnt1-1) shows a strong auxin excess phenotype and are allelic to sur-2. CYP83A1 is the closest relative to CYP83B1 and shares 63% amino acid sequence identity. Although expression of CYP83A1 under control of its endogenous promoter in the rnt1-1 background does not prevent the auxin excess and indole glucosinolate deficit phenotype caused by the lack of the CYP83B1 gene, ectopic overexpression of CYP83A1 using a 35S promoter rescues the rnt1-1 phenotype. CYP83A1 and CYP83B1 heterologously expressed in yeast (Saccharomyces cerevisiae) cells show marked differences in their substrate specificity. Both enzymes convert indole-3-acetaldoxime to a thiohydroximate adduct in the presence of NADPH and a nucleophilic thiol donor. However, indole-3-acetaldoxime has a 50-fold higher affinity toward CYP83B1 than toward CYP83A1. Both enzymes also metabolize the phenylalanine- and tyrosine-derived aldoximes. Enzyme kinetic comparisons of CYP83A1 and CYP83B1 show that indole-3-acetaldoxime is the physiological substrate for CYP83B1 but not for CYP83A1. Instead, CYP83A1 catalyzes the initial conversion of aldoximes to thiohydroximates in the synthesis of glucosinolates not derived from tryptophan. The two closely related CYP83 subfamily members therefore are not redundant. The presence of putative auxin responsive cis-acting elements in the CYP83B1 promoter but not in the CYP83A1 promoter supports the suggestion that CYP83B1 has evolved to selectively metabolize a tryptophan-derived aldoxime intermediate shared with the pathway of auxin biosynthesis in Arabidopsis.

Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana.

Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone.

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.

CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis.

Auxins are growth regulators involved in virtually all aspects of plant development. However, little is known about how plants synthesize these essential compounds. We propose that the level of indole-3-acetic acid is regulated by the flux of indole-3-acetaldoxime through a cytochrome P450, CYP83B1, to the glucosinolate pathway. A T-DNA insertion in the CYP83B1 gene leads to plants with a phenotype that suggests severe auxin overproduction, whereas CYP83B1 overexpression leads to loss of apical dominance typical of auxin deficit. CYP83B1 N-hydroxylates indole-3-acetaldoxime to the corresponding aci-nitro compound, 1-aci-nitro-2-indolyl-ethane, with a K(m) of 3 microM and a turnover number of 53 min(-1). The aci-nitro compound formed reacts non-enzymatically with thiol compounds to produce an N-alkyl-thiohydroximate adduct, the committed precursor of glucosinolates. Thus, indole-3-acetaldoxime is the metabolic branch point between the primary auxin indole-3-acetic acid and indole glucosinolate biosynthesis in Arabidopsis.

Overproduction of a P450 that metabolizes diazinon is linked to a loss-of-function in the chromosome 2 ali-esterase (MdalphaE7) gene in resistant house flies.

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of trans-regulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alphaE7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation in alphaE7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alphaE7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon.

Functional interactions in cytochrome P450BM3. Evidence that NADP(H) binding controls redox potentials of the flavin cofactors.

NADP(H) binding is essential for fast electron transfer through the flavoprotein domain of the fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized and partially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors and electron transfer. Measurements by three different approaches demonstrated a relatively low affinity of oxidized P450BM3 for NADP(+), with a K(d) of about 10 microM. NADPH binding is also relatively weak (K(d) approximately 10 microM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electron reduced enzyme binds NADP(+) with a K(d) in the submicromolar range. NADP(H) binding induces conformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescence quenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalytically competent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well as the interflavin electron transfer steps, is readily reversible, as demonstrated by a hydride ion exchange (transhydrogenase) reaction between NADPH and NADP(+) or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor required for the transhydrogenase activity. The equilibrium constants of each electron transfer step of the flavoprotein domain during catalytic turnover have been calculated. The values obtained differ from those calculated from equilibrium redox potentials by as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).

Inducible P450s of the CYP9 family from larval Manduca sexta midgut.

Several related cytochrome P450 cDNAs belonging to the CYP9 family have been cloned from the midgut of larval tobacco hornworms, Manduca sexta. The first P450, CYP9A2, was obtained by RT-PCR using degenerate primers. Northern blot analysis of expression in the midgut using the CYP9A2 probe revealed a significant induction by a variety of chemicals. Diets supplemented with the wild tomato compound 2-undecanone caused a dose-dependent induction which peaked after 48 h. Induction was also observed after addition to the diet of indole-3-carbinol, phenobarbital, 2-tridecanone and xanthotoxin. Neither alpha-pinene, clofibrate nor nicotine were effective inducers. The CYP9A2 probe hybridized to two mRNA species, one of 2. 0 kb and another of 4.2 kb, suggesting cross-hybridization to other P450 mRNAs. Additional P450 clones of the CYP9 family were then obtained and sequenced. Northern hybridization revealed that the 4.2 kb band also hybridized to CYP9A4 whereas the 2.0 kb hybridized to CYP9A5. Despite being 91% identical, CYP9A4 and CYP9A5 were induced differentially by clofibrate and xanthotoxin. Multiple P450 genes from various families are therefore induced in Lepidoptera in response to plant allelochemicals or xenobiotics.

Intron-exon organization and phylogeny in a large superfamily, the paralogous cytochrome P450 genes of Arabidopsis thaliana.

The cytochrome P450 gene superfamily is represented by 80 genes in animal genomes and perhaps more than 300 genes in plant genomes. We analyzed about half of all Arabidopsis P450 genes, a very large dataset of truly paralogous genes. Sequence alignments were used to draw phylogenetic trees, and this information was compared with the intron-exon organization of each P450 gene. We found 60 unique intron positions, of which 37 were phase 0 introns. Our results confirm the polyphyletic origin of plant P450 genes. One group of these genes, the A-type P450s, are plant specific and characterized by a simple organization, with one highly conserved intron. Closely related A-type P450 genes are often clustered in the genome with as many as a dozen genes (e.g., of the CYP71 subfamily) on a short stretch of chromosome. The other P450 genes (non-A-type) form several distinct clades and are characterized by numerous introns. One such clade contains the two CYP51 genes, which are thought to encode obtusifoliol 14a demethylase. The two CYP51 genes have a single intron that is not shared with CYP51 genes from vertebrates or fungi, or with any other Arabidopsis P450 gene. Only a few of the Arabidopsis P450 genes are intronless (e.g., the CYP710A and CYP96A subfamilies). There was a relatively good correlation between intron conservation and phylogenetic relationships between members of the P450 subfamilies. Gene organization appears to be a useful tool in establishing the evolutionary relatedness of P450 genes, which may help in predictions of P450 function.

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues.

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.

Terpenoid omega-hydroxylase (CYP4C7) messenger RNA levels in the corpora allata: a marker for ovarian control of juvenile hormone synthesis in Diploptera punctata.

Ribonuclease protection assays were used to measure changes in allatal transcript levels of the CYP4C7 gene which encodes a cytochrome P450 terpenoid omega-hydroxylase thought to play a role in the metabolism of JH and its precursors. Denervation of the corpora allata does not affect the pattern of expression of the CYP4C7 gene. Transplantation experiments show that CYP4C7 mRNA levels are dependent on a humoral factor characteristic of the reproductive state of the insect. Messenger RNA levels rise substantially in mated or denervated females, or in mated or virgin females treated with hydroprene, when the follicle length is over 1.5 mm. Vitellogenic ovaries however exert a negative influence on CYP4C7 expression, as ovariectomy in mated females causes a premature rise in CYP4C7 mRNA levels. The half-life of the CYP4C7 transcript is approx. 2 h when the corpora allata are incubated in vitro. Under these conditions, coincubation with a post-vitellogenic ovary maintains high CYP4C7 transcript levels in the glands. Excess juvenile hormone or analog applied at the end of vitellogenesis blocks ovulation or causes abortion of embryos deposited in the brood sac. We conclude that expression of the CYP4C7 gene is tightly controlled by the ovary, and it coincides with the ovarian signal to turn off juvenile hormone synthesis. The role of the CYP4C7 enzyme may be to ensure the clearance of allatal juvenile hormone and its precursors at the end of the gonotrophic cycle.

Cytochromes P450: a success story.

SUMMARY: Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.