Biomedical subjects

Mary A Schuler

Publications and source records attributed to Mary A Schuler.

At least 19 recordsLinked to original sources

PubMed2007

Allelochemical induction of cytochrome P450 monooxygenases and amelioration of xenobiotic toxicity in Helicoverpa zea.

Polyphagous herbivores encounter allelochemicals as complex mixtures in their host plants, and the toxicity of an individual compound may be influenced by the chemical matrix in which it is encountered. Certain plant constituents may reduce toxicity of cooccurring compounds by inducing detoxification systems, including cytochrome P450s, which can metabolize a broad range of substances. The polyphagous corn earworm Helicoverpa zea encounters a diversity of plant allelochemicals in its many host plants and, as well, can encounter aflatoxins, mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus that infect damaged grains. Dietary supplementation of each of three plant allelochemicals that are frequently (coumarin, COU), occasionally (indole-3-carbinol, 13C), or rarely (xanthotoxin, XAN) encountered by H. zea larvae substantially reduced the toxicity of aflatoxin B1 (AFB1) to H. zea. Compared to fourth instars on diets containing 1 microg/g AFB1 that failed to develop and pupate, fourth instars on diets containing I3C and XAN increased in mass by 216.1 and 700% after 6 days, and pupated at rates of 40 and 88%, respectively. Diets containing COU or XAN also significantly reduced the mortality rates of caterpillars exposed to the insecticides, diazinon and carbaryl. Diets containing COU and XAN increased CYP6B8 transcripts 2.6-fold; CYP321A1 transcripts increased 20.7, 8.3, and 10.6-fold in response to COU, I3C, and XAN, respectively. These results indicate that consumption of plant allelochemicals can ameliorate toxicity of natural and synthetic toxins encountered by insects, and they suggest that P450s induced by these allelochemicals contribute to detoxification of these chemicals in H. zea.

Animals↗

PubMed2007

Molecular mechanisms of metabolic resistance to synthetic and natural xenobiotics.

Xenobiotic resistance in insects has evolved predominantly by increasing the metabolic capability of detoxificative systems and/or reducing xenobiotic target site sensitivity. In contrast to the limited range of nucleotide changes that lead to target site insensitivity, many molecular mechanisms lead to enhancements in xenobiotic metabolism. The genomic changes that lead to amplification, overexpression, and coding sequence variation in the three major groups of genes encoding metabolic enzymes, i.e., cytochrome P450 monooxygenases (P450s), esterases, and glutathione-S-transferases (GSTs), are the focus of this review. A substantial number of the adaptive genomic changes associated with insecticide resistance that have been characterized to date are transposon mediated. Several lines of evidence suggest that P450 genes involved in insecticide resistance, and perhaps insecticide detoxification genes in general, may share an evolutionary association with genes involved in allelochemical metabolism. Differences in the selective regime imposed by allelochemicals and insecticides may account for the relative importance of regulatory or structural mutations in conferring resistance.

Animals↗

PubMed2006

The cytochrome P450 gene family CYP157 does not contain EXXR in the K-helix reducing the absolute conserved P450 residues to a single cysteine.

In this work, we have spectroscopically characterised CYP157C1 from Streptomyces coelicolor A3(2) which has the motif E(297)QSLW(301) rather than the invariant EXXR motif in the P450 K-helix. Site-directed mutagenesis of native E(297)QSLW(301) in CYP157C1 to E(297)ESLR(301) or E(297)QSRW(301) both containing standard EXXR motifs produced cytochrome P420 proteins thought to be inactive forms of P450 even though wild type CYP157C1 has the spectral properties of a normal P450. These results indicate that the EXXR motif is not required in all CYP tertiary architectures and only a single cysteine residue, which coordinates as the fifth thiolate ligand to the P450 haem iron, is invariant in all CYPs structures.

Amino Acid Motifs↗

PubMed2006

CYP6B1 and CYP6B3 of the black swallowtail (Papilio polyxenes): adaptive evolution through subfunctionalization.

Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6B1 and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.

Adaptation, Biological↗

PubMed2006

Class-dependent sequence alignment strategy improves the structural and functional modeling of P450s.

Different procedures for obtaining homology models for P450s are investigated using various sequence alignments sharing various levels of sequence identity with available P450 crystal structures. In this analysis, we have investigated how well homology modeling can reproduce known crystal structures as well as how effectively these homology models can be used to reproduce known ligand-binding modes. Homology models obtained from sequence alignments that discriminate between Class I and Class II P450s are significantly closer to the experimental crystal structures and more closely reproduce known ligand's binding modes, than those obtained using sequence alignments that combine Class I and Class II P450s. The quality of the models is slightly improved by constructing hybrid-structure models that model three of the most variable regions of P450s independently from the rest of the protein: the B region that includes SRS1, the FG region that includes SRS2 and SRS3 and the beta4 region that includes SRS6.

Animals↗

PubMed2006

Toxicity of aflatoxin B1 to Helicoverpa zea and bioactivation by cytochrome P450 monooxygenases.

Infestation of corn (Zea mays) by corn earworm (Helicoverpa zea) predisposes the plant to infection by Aspergillus fungi and concomitant contamination with the carcinogenic mycotoxin aflatoxin B1 (AFB1). Although effects of ingesting AFB1 are well documented in livestock and humans, the effects on insects that naturally encounter this mycotoxin are not as well defined. Toxicity of AFB1 to different stages of H. zea (first, third, and fifth instars) was evaluated with artificial diets containing varying concentrations. Although not acutely toxic at low concentrations (1-20 ng/g), AFB1 had significant chronic effects, including protracted development, increased mortality, decreased pupation rate, and reduced pupal weight. Sensitivity varied with developmental stage; whereas intermediate concentrations (200 ng/g) caused complete mortality in first instars, this same concentration had no detectable adverse effects on larvae encountering AFB1 in fifth instar. Fifth instars consuming AFB1 at higher concentrations (1 microg/g), however, displayed morphological deformities at pupation. That cytochrome P450 monooxygenases (P450s) are involved in the bioactivation of aflatoxin in this species is evidenced by the effects of piperonyl butoxide (PBO), a known P450 inhibitor, on toxicity; whereas no fourth instars pupated in the presence of 1 mug/g AFB1 in the diet, the presence of 0.1% PBO increased the pupation rate to 71.7%. Pupation rates of both fourth and fifth instars on diets containing 1 mug/g AFB1 also increased significantly in the presence of PBO. Effects of phenobarbital, a P450 inducer, on AFB1 toxicity were less dramatic than those of PBO. Collectively, these findings indicate that, as in many other vertebrates and invertebrates, toxicity of AFB1 to H. zea results from P450-mediated metabolic bioactivation.

Aflatoxin B1↗

PubMed2006

Inhibition of CYP6B1-mediated detoxification of xanthotoxin by plant allelochemicals in the black swallowtail (Papilio polyxenes).

The structural and biosynthetic diversity of allelochemicals in plants is thought to arise from selection for additive toxicity as a consequence of toxin mixture or for enhanced toxicity as a result of synergism. In order to understand how insects cope with this type of plant defense, we tested the effects of some allelochemicals in host plants of the black swallowtail Papilio polyxenes on the xanthotoxin-metabolic activity of CYP6B1, the principal enzyme responsible for the detoxification of furanocoumarins in this caterpillar. Additionally, the effects of some synthetic compounds not normally encountered by P. polyxenes on CYP6B1 were tested. These studies demonstrate that the integrity of furanocoumarin structure is important for competitive binding to the active site of CYP6B1, even though the carbonyl group on the pyranone ring apparently does not affect its inhibitory capacity, as in the case of furanochromones. Angular furanocoumarins are generally less phototoxic to many organisms than linear furanocoumarins due to their reduced capacity for cross-linking DNA strands, yet they are more toxic than linear furanocoumarins to black swallowtail larvae. This enhanced toxicity in vivo may be due to the ability of angular furanocoumarins to bind to the active site of CYP6B1 without being rapidly metabolized. This binding reduces the availability of CYP6B1 to metabolize other linear furanocoumarins. The structure-activity relationships for methylenedioxyphenyl compounds, flavonoids, imidazole, and imidazole derivatives are also discussed in light of their capacity to inhibit the xanthotoxin-metabolic activity of CYP6B1.

Animals↗

PubMed2006

Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases.

Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence.

Animals↗

PubMed2005

Variations in CYP74B2 (hydroperoxide lyase) gene expression differentially affect hexenal signaling in the Columbia and Landsberg erecta ecotypes of Arabidopsis.

The CYP74B2 gene in Arabidopsis (Arabidopsis thaliana) ecotype Columbia (Col) contains a 10-nucleotide deletion in its first exon that causes it to code for a truncated protein not containing the P450 signature typical of other CYP74B subfamily members. Compared to CYP74B2 transcripts in the Landsberg erecta (Ler) ecotype that code for full-length hydroperoxide lyase (HPL) protein, CYP74B2 transcripts in the Col ecotype accumulate at substantially reduced levels. Consistent with the nonfunctional HPL open reading frame in the Col ecotype, in vitro HPL activity analyses using either linoleic acid hydroperoxide or linolenic acid hydroperoxide as substrates show undetectable HPL activity in the Col ecotype and C6 volatile analyses using leaf homogenates show substantially reduced amounts of hexanal and no detectable trans-2-hexenal generated in the Col ecotype. P450-specific microarrays and full-genome oligoarrays have been used to identify the range of other transcripts expressed at different levels in these two ecotypes potentially as a result of these variations in HPL activity. Among the transcripts expressed at significantly lower levels in Col leaves are those coding for enzymes involved in the synthesis of C6 volatiles (LOX2, LOX3), jasmonates (OPR3, AOC), and aliphatic glucosinolates (CYP83A1, CYP79F1, AOP3). Two of the three transcripts coding for aliphatic glucosinolates (CYP83A1, AOP3) are also expressed at significantly lower levels in Col flowers.

Aldehyde-Lyases↗

PubMed2005

Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades.

Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site. One such element is the xenobiotic response element to xanthotoxin (XRE-Xan) that lies upstream of consensus XRE-AhR (xenobiotic response element to the aryl hydrocarbon receptor) and OCT-1 (octamer-1 binding site) element known to be utilized in mammalian aryl hydrocarbon response cascades. Two-plasmid transfections conducted in Sf9 cells have indicated that XRE-Xan, XRE-AhR and a number of other proximal elements, but not OCT-1, are critical for basal as well as xanthotoxin- and benzo[alpha]pyrene-induced transcription of the CYP6B1 promoter. Four-plasmid transfections with vectors co-expressing the spineless (Ss) and tango (Tgo) proteins, the Drosophila melanogaster homologues of mammalian AhR and ARNT, have indicated that these proteins enhance basal expression of the CYP6B1 promoter but not the magnitude of its xanthotoxin and benzo[alpha]pyrene induction. Based on these results, we propose that these Drosophila transcription factors modulate basal expression of this promoter in a ligand-independent manner and attenuate its subsequent responses to planar aryl hydrocarbons (benzo[alpha]pyrene) and allelochemicals (xanthotoxin).

Animals↗

PubMed2005

Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel.

CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.

Amino Acid Sequence↗

PubMed2005

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803.

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.

Bacterial Proteins↗

PubMed2005

Differential expression and evolution of the Arabidopsis CYP86A subfamily.

Some members of the Arabidopsis (Arabidopsis thaliana) CYP86A and CYP94B cytochrome P450 monooxygenase subfamilies, which share some sequence homology with the animal and fungal fatty acid hydroxylases, have been functionally defined as fatty acid omega-hydroxylases. With these activities, these and other fatty acid hydroxylases have potential roles in the synthesis of cutin, production of signaling molecules, and prevention of accumulation of toxic levels of free fatty acids. The constitutive and stress-inducible patterns of the five Arabidopsis CYP86A subfamily members have been defined in 7-d-old seedlings and 1-month-old plant tissues grown under normal conditions, and 7-d-old seedlings treated with different hormones (indole-3-acetic acid, abscisic acid, gibberellin, methyl jasmonic acid, brassinosteroid, salicylic acid), chemicals (clofibrate, 1-aminocyclopropane-1 carboxylic acid), or environmental stresses (cold, wounding, drought, mannitol, etiolation). Very distinct expression patterns exist for each of these fatty acid hydroxylases under normal growth conditions and in response to environmental and chemical stresses. Analysis of the promoter sequences for each of these genes with their expression patterns has highlighted a number of elements in current databases that potentially correlate with the responses of individual genes.

Amino Acid Sequence↗

PubMed2005

Bicistronic and fused monocistronic transcripts are derived from adjacent loci in the Arabidopsis genome.

Comparisons of full-length cDNAs and genomic DNAs available for Arabidopsis thaliana described here indicate that some adjacent loci are transcribed into extremely long RNAs spanning two annotated genes. Once expressed, some of these transcripts are post-transcriptionally spliced within their coding and intergenic sequences to generate bicistronic transcripts containing two complete open reading frames. Others are spliced to generate monocistronic transcripts coding for fusion proteins with sequences derived from both loci. RT-PCR of several P450 transcripts in this collection indicates that these extended transcripts exist side by side with shorter monocistronic transcripts derived from the individual loci in each pair. The existence of these unusual transcripts highlights variations in the processes of transcription and splicing that could not possibly have been predicted in the algorithms used for genome annotation and splice site predictions.

Arabidopsis↗

PubMed2004

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea.

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.

Amino Acid Sequence↗

PubMed2004

Co-incorporation of heterologously expressed Arabidopsis cytochrome P450 and P450 reductase into soluble nanoscale lipid bilayers.

Heterologous expression of CYP73A5, an Arabidopsis cytochrome P450 monooxygenase, in baculovirus-infected insect cells yields correctly configured P450 detectable by reduced CO spectral analysis in microsomes and cell lysates. Co-expression of a housefly NADPH P450 reductase substantially increases the ability of this P450 to hydroxylate trans-cinnamic acid, its natural phenylpropanoid substrate. For development of high-throughput P450 substrate profiling procedures, membrane proteins derived from cells overexpressing CYP73A5 and/or NADPH P450 reductase were incorporated into soluble His(6)-tagged nanoscale lipid bilayers (Nanodiscs) using a simple self-assembly process. Biochemical characterizations of nickel affinity-purified and size-fractionated Nanodiscs indicate that CYP73A5 protein assembled into Nanodiscs in the absence of NADPH P450 reductase maintains its ability to bind its t-cinnamic acid substrate. CYP73A5 protein co-assembled with P450 reductase into Nanodiscs hydroxylates t-cinnamic acid using reduced pyridine nucleotide as an electron source. These data indicate that baculovirus-expressed P450s assembled in Nanodiscs can be used to define the chemical binding profiles and enzymatic activities of these monooxygenases.

Animals↗

PubMed2004

Structural and functional divergence of insect CYP6B proteins: From specialist to generalist cytochrome P450.

How polyphagous herbivores cope with the diversity and unpredictability of plant defenses remains largely unknown at both the genetic and molecular levels. To examine whether generalist counterdefense enzymes are structurally more flexible and functionally more diverse, two counterdefensive allelochemical-metabolizing cytochrome P450 proteins, CYP6B1 from the specialist Papilio polyxenes, feeding on furanocoumarin-containing plants, and CYP6B8 from the generalist Helicoverpa zea, feeding on hundreds of host plant species, are compared structurally and functionally. Molecular modeling indicates that CYP6B8 has more flexible overall folding, a more elastic catalytic pocket, and one more substrate access channel than CYP6B1. Baculovirus-mediated expression of the CYP6B8 and CYP6B1 proteins demonstrates that CYP6B8 metabolizes six biosynthetically diverse plant allelochemicals (xanthotoxin, quercetin, flavone, chlorogenic acid, indole-3-carbinol, and rutin) and three insecticides (diazinon, cypermethrin, and aldrin), whereas CYP6B1 metabolizes only two allelochemicals (xanthotoxin and flavone) and one insecticide (diazinon) of those tested. These results indicate that generalist counterdefense proteins are capable of accepting a more structurally diverse array of compounds compared with specialist counterdefense proteins.

Amino Acid Sequence↗

PubMed2004

Identification of variable amino acids in the SRS1 region of CYP6B1 modulating furanocoumarin metabolism.

The homology model of Papilio polyxenes CYP6B1 places Ile115, one of two variable amino acids, in the SRS1 of various CYP6B subfamily proteins in close proximity to the heme and Ala113, another variable amino acid, in a more distal position. We have constructed mutant CYP6B1 proteins altered at either of these positions and homology models of each based on multiple alignments with crystallized P450 proteins. The homology models suggest the existence of significant structural diversity in the hydrogen bond network surrounding the heme as a result of single point mutations in SRS1. Mutagenesis of Ile115 or Ala113 to other residues present in the insect CYP6B subfamily indicates that these amino acids control the spin state of the heme and, as a result, the catalytic activity of this monooxygenase. In particular, the I115L mutation significantly increases the spin state of the heme coordinately with 2- to 4-fold increases in its turnover of linear furanocoumarins. Other A113V, A113L, A113Q, and A113E mutations display more variation in their effects but, in each case, strong correlations exist between furanocoumarin turnover and heme spin state. These data demonstrate that variable amino acids in SRS1 of the insect CYP6B subfamily exert dramatic effects on the range of furanocoumarins metabolized, even when they occur in positions potentially distal from the substrate. These effects are possibly mediated through rearrangement of the local hydrogen bond network.

Amino Acid Sequence↗