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R Feyereisen

Publications and source records attributed to R Feyereisen.

At least 19 recordsLinked to original sources
PubMed1997

Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1.

Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.

Animals↗
PubMed1996

Functional interactions in cytochrome P450BM3. Fatty acid substrate binding alters electron-transfer properties of the flavoprotein domain.

P450BM3 is a bacterial fusion protein between a cytochrome P450 fatty acid hydroxylase (CYP102) and an FAD- and FMN-containing flavoprotein homologous to NADPH: cytochrome P450 reductase. It has been shown that incubation of P450BM3 with NADPH in the absence of a fatty acid substrate results in inhibition of hydroxylase activity [Narhi, L. O., & Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169]. We show that laurate-dependent oxidation of NADPH and oxygen consumption are also inhibited under those conditions. The inhibited enzyme is unable to transfer electrons to the heme iron, but reduces artificial electron acceptors such as cytochrome c, 2,6-dichlorophenolindophenol, or ferricyanide. Incubation with these acceptors rapidly restores hydroxylase activity of P450BM3. The active enzyme is able to catalyze the reduction of cytochrome c and hydroxylation of laurate simultaneously. Cytochrome c has no effect on the K(m) and Vmax of laurate hydroxylation. Laurate and other substrates stimulate cytochrome c reduction by 50-70%. Carbon monoxide inhibits hydroxylase activity, but stimulates cytochrome c reduction 3-4 fold and has no effect on the K(m) for cytochrome c. This stimulation requires binding of a substrate at the heme catalytic site. Laurate binding induces conformational changes in the flavoprotein domain as shown by a 2-fold increase of the flavin fluorescence. Inactivation of P450BM3 by NADPH abolishes the stimulation of cytochrome c reduction by laurate and CO. Complete inhibition of hydroxylase activity correlates with complete lack of stimulation of cytochrome c reduction. The results suggest that a specific conformation of the two domains is maintained in the active P450BM3, ensuring high hydroxylase activity. Cytochrome c reductase and hydroxylase activities of P450BM3 involve different sites of interaction with the flavoprotein domain, different catalytic intermediates, and different rate-limiting steps.

Bacterial Proteins↗
PubMed1996

Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b5.

A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced. The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5. The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates. NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.

Amino Acid Sequence↗
PubMed1996

Target of cockroach allatostatin in the pathway of juvenile hormone biosynthesis.

Allatostatins, peptides with an YXFGL-amide C-terminus, cause a rapid and reversible inhibition of the sesquiterpenoid juvenile hormone III biosynthesis by cockroach corpora allata in vitro. Inhibition is relieved by farnesol and by mevalonate, and neither HMG-CoA reductase nor HMG-CoA synthesis (HMG-CoA synthase and acetoacetyl-CoA lyase) are inhibited by allatostatin. The neuropeptide is more effective as an inhibitor of JH III synthesis in glucose or amino acid-driven glands than in acetate-driven glands. Incorporation of label from acetate into JH III is increased whereas incorporation of label from glucose is decreased by allatostatin. Citrate is a very effective precursor for HMG-CoA synthesis in homogenates of corpora allata. We propose that inhibition of JH III biosynthesis by the tridecapeptide APSGAQRLYGFGL-amide occurs at the first committed step(s) of JH III synthesis, i.e. the transfer of 2C units from mitochondria to the cytoplasm by the tricarboxylate carrier and/or the ATP-citrate lyase.

Acetyl Coenzyme A↗
PubMed1996

Cytochrome P450 gene clusters in Drosophila melanogaster.

Twelve cytochrome P450 cDNA fragments were cloned from Drosophila melanogaster by reverse transcriptase/PCR (RT/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes, Cyp4dl and Cyp4d2, have previously been described. In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of the X chromosome and the other on the left arm of chromosome 2. Interestingly the latter cluster comprises widely divergent genes belonging both to the CYP9 and CYP4 families and also to the CYP6 family (Cyp6a2). Putative allelic variants of several of the genes were found in different insecticide-resistant and -susceptible strains (Hikone R, Haag 79 and Oregon R). The identification of these genes and alleles will allow us to clarify the involvement of P450s in xenobiotic metabolism and will facilitate a genetic analysis of P450 functions in insects.

Amino Acid Sequence↗
PubMed1996

Molecular biology and evolution of resistance of toxicants.

To the prevailing biochemical/physiological classification of mechanisms of organismal resistance to toxicants, an additional molecular dimension is proposed. Predictions are developed regarding the relative prevalence of different classes of mutations and are found to compare favorably with reports from the literature. In particular, point mutations in target loci were the dominant form of resistance for both lab and field selection. Amplifications of target loci were less common than structural mutations, and more common for lab-selected than for field-selected strains. Amplification was the most common mechanism of up-regulation of metabolizing enzymes. In comparison, only one mutation involving cis-regulation and several involving trans-acting regulation were found. Mutations involving gene disruption and down-regulation were uncommon, but were found in appropriate cases, i.e., when toxicants stimulated rather than inhibited target function and when metabolizing enzymes converted toxicants into more toxic metabolites. Additional phenomena of likely but uncertain importance are genetic "succession," recombinational limitation, and negative cross-resistance. More work on these phenomena and on quantification of fitness costs of resistance is recommended.

Animal Population Groups↗
PubMed1996

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature.

We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995. These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species. Of 74 gene families so far described, 14 families exist in all mammals examined to date. These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively. Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions. Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient. One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes. This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described. For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), representing "cytochrome P450', be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen is no longer recommended in mouse gene nomenclature. "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued. If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included. The human nomenclature system should be used for all species other than mouse and Drosophila. The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens. This nomenclature system is similar to that proposed in our previous updates.

Alleles↗
PubMed1995

Expression of cytochrome P450 genes of the CYP4 family in midgut and fat body of the tobacco hornworm, Manduca sexta.

Two conserved regions in the alignment of cytochrome P450 family 4 (CYP4) proteins served as guide to the synthesis of degenerate oligonucleotide primers. The primers were used in PCR from a midgut cDNA library and RT-PCR from fat body mRNA, both from last instar larvae of the tobacco hornworm, Manduca sexta. The PCR products of 443-449 bp were cloned and sequenced. Nine P450 clones representing four new genes were obtained from the midgut. Fifteen P450 clones representing three new genes were obtained from the fat body. Two genes were expressed in both tissues. A number of putative allelic variants were also observed for three of the P450 genes. The resulting sequences of 130-132 amino acids were aligned to generate a parsimony analysis of CYP4 P450 proteins. Two new subfamilies of CYP4 were designated from M. sexta by these procedures, CYP4L and CYP4M. The sequence of a full-length cDNA clone for CYP4M2 (41.2% identity to CYP4C1) confirmed that the PCR products obtained by this method were P450s belonging to the CYP4 family. The developmental expression of the CYP4 genes appeared to be coordinately regulated in both fat body and midgut. In the fat body, CYP4 mRNA levels declined after the first day of the final larval instar, peaked during the wandering stage, and fell again until the prepupal molt. Midgut CYP4 mRNA levels were higher during the active feeding, midwandering, prepupal, and pupal stages. Addition of 2-tridecanone or 2-undecanone to the diet induced several P450s in the midgut and in the fat body. Phenobarbital induced CYP4M1 in the fat body and dietary clofibrate induced the mRNA levels of CYP4M1 and CYP4M3 in the midgut. The results indicate that at least four CYP4 genes are expressed in single tissues of a Lepidopteran insect. Several of these P450 may be involved in tissue responses to xenobiotics.

Adipose Tissue↗
PubMed1995

Molecular biology of insecticide resistance.

The widespread use of insecticides has amounted to a large scale 'experiment' in natural selection of insects by chemicals of toxicological importance to humans. Specific examples in which the molecular basis of insecticide resistance has been studied in detail are presented here. The biochemical/physiological mechanisms of resistance can be categorized as target site insensitivity, increased metabolic detoxification and sequestration or lowered availability of the toxicant. These are achieved at the molecular level by: point mutations in the ion channel portion of a GABA receptor subunit (cyclodiene insecticides); point mutations in the vicinity of the acetylcholinesterase (AChE) active site (organophosphorus and carbamate insecticide resistance); amplification of esterase genes (organophosphorus and carbamate insecticides); mutations linked genetically to a sodium channel gene (DDT and pyrethroid insecticides); and yet uncharacterized mutations leading to the up-regulation of detoxification enzymes, such as cytochrome P450 and glutathione S-transferases (many classes of insecticides). In several cases, the selection of a precisely homologous mutation has been observed in different insect species.

Animals↗
PubMed1995

Glutathione S-transferases from larval Manduca sexta midgut: sequence of two cDNAs and enzyme induction.

Two glutathione S-transferase (GST) clones from a larval midgut cDNA library of the tobacco hornworm, Manduca sexta were sequenced. The nucleotide sequence of the first clone, M. sexta GST1, encoded a protein of 217 amino acids with a predicted molecular weight of 24,644 and isoelectric point of 4.8. The M. sexta GST1 was 45.9-48.6% identical to GSTs from Musca domestica and several Drosophila species. The M. sexta GST2 cDNA encoded a protein of 203 amino acids with a predicted molecular weight of 23,596 and isoelectric point of 5.5. The M. sexta GST2 shared 44.8-50.0% sequence identity to a second cluster of insect GSTs from M. domestica, D. melanogaster and Anopheles gambiae. GST1 and GST2 were only 24.1% identical in amino acid sequence. The divergence of these two classes of insect GSTs occurred before the radiation of Diptera and Lepidoptera. Northern analysis of the expression of these GSTs showed increased GST1 mRNA levels in midguts of larvae fed diets containing 2-undecanone, or phenobarbital. Midgut and fat body cytosolic GST activities were induced when larvae were fed diets containing 2-tridecanone, 2-undecanone, or phenobarbital. Partial purification of midgut GSTs by size-exclusion and glutathione affinity chromatography resulted in a series of isoelectric focusing bands, with the major one corresponding to the predicted isoelectric point of the M. sexta GST1. In summary, two midgut GSTs have been identified on the basis of cDNA sequence and one of these, GST1, was inducible by dietary chemicals.

Amino Acid Sequence↗
PubMed1995

Photoaffinity labeling of methyl farnesoate epoxidase in cockroach corpora allata.

The last enzyme in the biosynthetic pathway to juvenile hormone III in the corpora allata of hemimetabolous insects is methyl farnesoate epoxidase, a cytochrome P450 monooxygenase. Assays with intact glands incubated in vitro and with gland homogenates have identified a series of 1,5-disubstituted imidazoles as potent inhibitors of the enzyme. We have designed, synthesized and tested two imidazoles, diazirine-Ice T and benzophenone-Ice T, in which a radiolabeled and photoactivatable diazirine or benzophenone group was introduced to label the hydrophobic substrate binding site of the enzyme. Our results show that these bifunctional compounds inhibit JH III synthesis by intact glands as well as methyl farnesoate epoxidation by gland homogenates. Moreover both compounds selectively label a protein of ca. 55 kDa in corpora allata of the cockroach, Diploptera punctata. These photoaffinity labels, which use an imidazole to coordinate to the heme iron and a photoreactive group to modify the hydrophobic substrate binding pocket, are specific and effective probes for the molecular analysis of methyl farnesoate epoxidase.

Affinity Labels↗
PubMed1995

A cluster of cytochrome P450 genes of the CYP6 family in the house fly.

A cluster of genes of the CYP6 family was found in a series of overlapping lambda DASH clones from a genomic library of the house fly, Musca domestica. Four complete genes, CYP6A3, CYP6A4, CYP6A5, and CYP6C1, and fragments of two other genes, CYP6A6 and CYP6C2, were closely linked on a 24-kb segment of DNA. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified segments of two of the genes showed that the cluster is localized on chromosome V of the house fly. Each gene contained a short intron of 57 to 125 bp interrupting a conserved Glu codon, as in the previously described CYP6A1 gene. The gene fragment CYP6A6 consisted only of the coding region downstream from this intron, i.e., about one-third of the complete P450. The gene fragment CYP6C2 was missing a short amino-terminal part of the coding region, and may represent the two last exons of a larger gene. Gene duplication and chromosomal inversion events may explain the origin of this cluster. The P450 proteins deduced from the nucleotide sequences shared 39-71% amino acid identity with each other. This low identity and the lack of evidence of recent gene conversion events suggested that this cluster may be evolutionarily ancient and that homologous clusters may be found in other holometabolous insects. Evidence for transcription of the genes and for correct splicing of the introns was obtained by northern blotting and reverse transcription polymerase chain reaction (RT-PCR) experiments. No overexpression was observed in any of three insecticide-resistant house fly strains. RT-PCR and sequencing also revealed the existence of other genes or alleles closely related to the members of this cluster.

Amino Acid Sequence↗
PubMed1994

Diversity of cytochrome P450 genes in the mosquito, Anopheles albimanus.

Degenerate oligonucleotide primers were designed for conserved regions of cytochrome P450 proteins of the CYP4 family and were used to amplify cDNA or genomic DNA from different strains of the New World malaria vector, Anopheles albimanus (Weidemann). The PCR products were cloned, sequenced and compared to each other and to members of the P450 family CYP4. Seventeen new P450 genes were identified in five CYP4 subfamilies. Five of the ten PCR products of genomic DNA were shown to contain a short (60-79 bp) intron at the same position as introns in the Drosophila CYP4D2 and CYP4E1 genes.

Amino Acid Sequence↗
PubMed1994

Expression of the allatostatin gene in endocrine cells of the cockroach midgut.

Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYVSEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several allatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.

Amino Acid Sequence↗
PubMed1994

Structure and chromosomal localization of CYP6A1, a cytochrome P450-encoding gene from the house fly.

We determined the sequence of a cytochrome P450-encoding gene, CYP6A1, in an insecticide-resistant strain (Rutgers) and in insecticide-susceptible strains (aabys and sbo) of the house fly Musca domestica. The deduced amino acid (aa) sequence of CYP6A1 is 98% identical between Rutgers and aabys, and it is identical between Rutgers and sbo. Differences in aa sequence occur in regions that are not thought to participate in the active site. CYP6A1 has a single intron, 60 bp in length. It does not occur in a position homologous to intron sites in genes of the related CYP3 family or other vertebrate P450 families. We mapped the transcription start point (tsp), TATA box and barbie box [Shaw and Fulco, J. Biol. Chem. 269 (1993) 2997-3004] of this phenobarbital-inducible gene. Although overexpression of CYP6A1 in strain Rutgers is controlled by a locus on chromosome II [Cariño et al., Insect Biochem. Mol. Biol. 24 (1994) 411-418], we have localized CYP6A1 to chromosome V, thus showing that a trans-acting factor controls the overexpression of the gene in the Rutgers strain.

Amino Acid Sequence↗
PubMed1994

Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450 system.

The house fly (Musca domestica) cytochrome P450 gene CYP6A1 was expressed in Escherichia coli. The native protein was produced at a level of 0.25-0.34 mumol/L (15-20 mg/L) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized spectrally and purified by a combination of hydrophobic interaction and hydroxyapatite chromatography. The house fly NADPH-cytochrome P450 reductase gene was also expressed in E. coli. Expression of a cytoplasmically directed reductase resulted in a protein that reduced cytochrome c but did not support P450 monooxygenase reactions. However, a periplasmically directed reductase was found to support monooxygenase reactions with CYP6A1 in a reconstituted system. The reconstituted system was effective in the epoxidation of the cyclodiene insecticides aldrin and heptachlor, with turnover rates of 12 and 34 min-1, respectively. The enzyme showed little detectable activity in the O-dealkylation and N-dealkylation of various compounds that are metabolized by house fly microsomes. Incubation with polyclonal antisera raised against purified CYP6A1 inhibited the microsomal epoxidation of heptachlor by 65%. Under the same conditions, the metabolism of 7-methoxy-4-methylcoumarin was inhibited only slightly. The results suggest that CYP6A1 is a major cyclodiene epoxidase in the house fly and that multiple P450 forms are responsible for the elevated monooxygenase activities in insecticide-resistant flies.

Animals↗
PubMed1994

Constitutive overexpression of the cytochrome P450 gene CYP6A1 in a house fly strain with metabolic resistance to insecticides.

Messenger RNA levels of the cytochrome P450 gene CYP6A1 were measured in the insecticide resistant Diazinon-R 'Rutgers' strain and in the susceptible strain sbo of the house fly with a cloned cDNA probe. The constitutive expression of the CYP6A1 gene was at least 10 times higher in the Rutgers strain than in the sbo strain. In both strains, CYP6A1 was inducible by phenobarbital treatment of the flies. Analysis of genomic DNA from the two strains indicated that there was no amplification of the CYP6A1 gene in the Rutgers strain. A developmental analysis revealed that CYP6A1 is expressed larvae and adults, and very low levels of CYP6A1 mRNA were detected in eggs and pupae. The constitutive overexpression of CYP6A1 in the Rutgers strain was observed in both larvae and adults. Crosses between the Rutgers strain and the multiply marked sbo strain revealed that the high constitutive expression of CYP6A1 in the Rutgers strain is controlled by one or more loci located on chromosome II.

Animals↗