RESUMO
Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C-C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C-C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C-C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C-C bond scission of loganic acid; Asp332Glu in the acid-alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.
Assuntos
Camptotheca , Domínio CatalíticoRESUMO
The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.
Assuntos
Liases , Humanos , Domínio Catalítico , Catálise , Simulação de Dinâmica MolecularRESUMO
Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.
Assuntos
Camptotheca , Sistema Enzimático do Citocromo P-450 , Glucosídeos Iridoides , Iridoides , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Camptotheca/enzimologia , Camptotheca/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Histidina/química , Histidina/genética , Glucosídeos Iridoides/metabolismo , Iridoides/metabolismo , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Triptaminas/metabolismoRESUMO
Divergent terpene indole alkaloid (TIA) pathways in Catharanthus roseus and Camptotheca acuminata generate vinblastine and vincristine, and camptothecin, respectively. In contrast to Catharanthus which feeds secologanin (from methylated loganin) into its species-specific late pathway, Camptotheca feeds secologanic acid (from unmethylated loganic acid) into its late pathway. Having identified putative Camptotheca secologanic acid synthases (SLASs) and cytochrome P450 reductases (CPRs) in transcriptome databases, we have demonstrated that two P450s, CYP72A564 and CYP72A565, are capable of utilizing both loganic acid and loganin to generate secologanic acid and secologanin. We have extended the previous report of these activities by CYP72A565 and CYP72A610 (Yang et al., 2019) by demonstrating that both Arabidopsis CPRs (ATR1, ATR2) couple with these CYP72A proteins in yeast microsomal assays and that purified Camptotheca CPR1 couples with them in in vitro reconstitution assays. Kinetic analyses of purified full-length Camptotheca SLASs have indicated that both process loganic acid with nearly identical catalytic rates and efficiencies as measured by their kcat and kcat/KM. In contrast, CYP72A564 processes loganin with two-fold greater efficiency than CYP72A565 correlating with the former's 3-fold greater affinity for loganin. The closely-related CYP72A730 does not bind or process either compound. Molecular modeling of these three proteins and comparisons with Catharanthus secologanin synthase (SLS) have identified key differences that likely determine their SLAS versus SLS selectivities. Our ability to reconstitute these SLAS/SLS activities provides valuable tools for further examinations of the residues involved in substrate recognition and determinations of their unusual mechanism of C-C bond scission.
Assuntos
Camptotheca , Catharanthus , Camptotecina , Alcaloides Indólicos , TerpenosRESUMO
The interactions between lipids and proteins are one of the most fundamental processes in living organisms, responsible for critical cellular events ranging from replication, cell division, signaling, and movement. Enabling the central coupling responsible for maintaining the functionality of the breadth of proteins, receptors, and enzymes that find their natural home in biological membranes, the fundamental mechanisms of recognition of protein for lipid, and vice versa, have been a focal point of biochemical and biophysical investigations for many decades. Complexes of lipids and proteins, such as the various lipoprotein factions, play central roles in the trafficking of important proteins, small molecules and metabolites and are often implicated in disease states. Recently an engineered lipoprotein particle, termed the nanodisc, a modified form of the human high density lipoprotein fraction, has served as a membrane mimetic for the investigation of membrane proteins and studies of lipid-protein interactions. In this review, we summarize the current knowledge regarding this self-assembling lipid-protein complex and provide examples for its utility in the investigation of a large number of biological systems.
Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Proteínas de Membrana/química , Nanoestruturas/química , Animais , Humanos , Lipoproteínas/químicaRESUMO
Loganin is an iridoid glycoside of interest as both an intermediate in the biosynthesis of indole alkaloids in plants and as a bioactive compound itself. Loganic acid methyltransferase catalyzes the methylation of a monoterpenoid glycoside precursor to produce loganin and demonstrates stereospecificity for the (6S,7R) substrate. We have biochemically characterized this biocatalyst and elucidated the basis for its strict substrate specificity. These studies could help facilitate the design of new classes of monoterpenoid indole alkaloids of pharmaceutical interest.
Assuntos
Glicosídeos Iridoides/metabolismo , Iridoides/metabolismo , Metiltransferases/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Metilação , Metiltransferases/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Across insect genomes, the size of the cytochrome P450 monooxygenase (CYP) gene superfamily varies widely. CYPome size variation has been attributed to reciprocal adaptive radiations in insect detoxification genes in response to plant biosynthetic gene radiations driven by co-evolution between herbivores and their chemically defended hostplants. Alternatively, variation in CYPome size may be due to random "birth-and-death" processes, whereby exponential increase via gene duplications is limited by random decay via gene death or transition via divergence. We examined CYPome diversification in the genomes of seven Lepidoptera species varying in host breadth from monophagous (Bombyx mori) to highly polyphagous (Amyelois transitella). CYPome size largely reflects the size of Clan 3, the clan associated with xenobiotic detoxification, and to some extent phylogenetic age. Consistently across genomes, families CYP6, CYP9 and CYP321 are most diverse and CYP6AB, CYP6AE, CYP6B, CYP9A and CYP9G are most diverse among subfamilies. Higher gene number in subfamilies is due to duplications occurring primarily after speciation and specialization ("P450 blooms"), and the genes are arranged in clusters, indicative of active duplicating loci. In the parsnip webworm, Depressaria pastinacella, gene expression levels in large subfamilies are high relative to smaller subfamilies. Functional and phylogenetic data suggest a correlation between highly dynamic loci (reflective of extensive gene duplication, functionalization and in some cases loss) and the ability of enzymes encoded by these genes to metabolize hostplant defences, consistent with an adaptive, nonrandom process driven by ecological interactions.
Assuntos
Evolução Biológica , Sistema Enzimático do Citocromo P-450/genética , Mariposas/enzimologia , Filogenia , Animais , Genoma de Inseto , Mariposas/classificação , Mariposas/genética , TranscriptomaRESUMO
Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.
Assuntos
Abelhas/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Fungicidas Industriais/toxicidade , Proteínas de Insetos/química , Nitrilas/toxicidade , Quercetina/antagonistas & inibidores , Triazóis/toxicidade , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/biossíntese , Animais , Abelhas/genética , Abelhas/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Fungicidas Industriais/química , Regulação da Expressão Gênica , Mel/análise , Inativação Metabólica/efeitos dos fármacos , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Simulação de Acoplamento Molecular , Nitrilas/química , Fosforilação Oxidativa/efeitos dos fármacos , Pólen/química , Pólen/metabolismo , Quercetina/biossíntese , Quercetina/química , Triazóis/químicaRESUMO
In the eusocial honey bee Apis mellifera, with reproductive queens and sterile workers, a female larva's developmental fate depends on its diet; nurse bees feed queen-destined larvae exclusively royal jelly, a glandular secretion, but worker-destined larvae receive royal jelly for 3 days and subsequently jelly to which honey and beebread are added. RNA-Seq analysis demonstrated that p-coumaric acid, which is ubiquitous in honey and beebread, differentially regulates genes involved in caste determination. Rearing larvae in vitro on a royal jelly diet to which p-coumaric acid has been added produces adults with reduced ovary development. Thus, consuming royal jelly exclusively not only enriches the diet of queen-destined larvae but also may protect them from inhibitory effects of phytochemicals present in the honey and beebread fed to worker-destined larvae.
RESUMO
In Candida albicans, the ERG11 gene encodes lanosterol demethylase, the target of the azole antifungals. Mutations in ERG11 that result in an amino acid substitution alter the abilities of the azoles to bind to and inhibit Erg11, resulting in resistance. Although ERG11 mutations have been observed in clinical isolates, the specific contributions of individual ERG11 mutations to azole resistance in C. albicans have not been widely explored. We sequenced ERG11 in 63 fluconazole (FLC)-resistant clinical isolates. Fifty-five isolates carried at least one mutation in ERG11, and we observed 26 distinct positions in which amino acid substitutions occurred. We mapped the 26 distinct variant positions in these alleles to four regions in the predicted structure for Erg11, including its predicted catalytic site, extended fungus-specific external loop, proximal surface, and proximal surface-to-heme region. In total, 31 distinct ERG11 alleles were recovered, with 10 ERG11 alleles containing a single amino acid substitution. We then characterized 19 distinct ERG11 alleles by introducing them into the wild-type azole-susceptible C. albicans SC5314 strain and testing them for susceptibilities to FLC, itraconazole (ITC), and voriconazole (VRC). The strains that were homozygous for the single amino acid substitutions Y132F, K143R, F145L, S405F, D446E, G448E, F449V, G450E, and G464S had a ≥ 4-fold increase in FLC MIC. The strains that were homozygous for several double amino acid substitutions had decreased azole susceptibilities beyond those conferred by any single amino acid substitution. These findings indicate that mutations in ERG11 are prevalent among azole-resistant clinical isolates and that most mutations result in appreciable changes in FLC and VRC susceptibilities.
Assuntos
Inibidores de 14-alfa Desmetilase/uso terapêutico , Azóis/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Esterol 14-Desmetilase/genética , Substituição de Aminoácidos , Antifúngicos/uso terapêutico , Candidíase/microbiologia , Domínio Catalítico/genética , Farmacorresistência Fúngica , Fluconazol/uso terapêutico , Humanos , Itraconazol/uso terapêutico , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Voriconazol/uso terapêuticoRESUMO
Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management.
Assuntos
Adaptação Fisiológica , Sistema Enzimático do Citocromo P-450/fisiologia , Insetos/fisiologia , Toxinas Biológicas/fisiologia , Animais , Sistema Enzimático do Citocromo P-450/química , Modelos MolecularesRESUMO
As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by â¼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses.
Assuntos
Abelhas/imunologia , Colapso da Colônia/induzido quimicamente , Ácidos Cumáricos/farmacologia , Regulação da Expressão Gênica/imunologia , Mel/análise , Inativação Metabólica/imunologia , Intoxicação por Organofosfatos/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Abelhas/genética , Cromatografia Líquida de Alta Pressão , Cumafos/toxicidade , Ácidos Cumáricos/química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Flavanonas/química , Flavanonas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Inativação Metabólica/genética , Pólen/química , Propionatos , Própole/química , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
Nanodiscs are self-assembled discoidal fragments of lipid bilayers 8-16 nm in diameter, stabilized in solution by two amphipathic helical scaffold proteins. As stable and highly soluble membrane mimetics with controlled lipid composition and ability to add affinity tags to the scaffold protein, nanodiscs represent an attractive model system for solubilization, isolation, purification, and biophysical and biochemical studies of membrane proteins. In this chapter we overview various approaches to structural and functional studies of different classes of integral membrane proteins such as ion channels, transporters, GPCR and other receptors, membrane enzymes, and blood coagulation cascade proteins which have been incorporated into nanodiscs. We outline the advantages provided by homogeneity, ability to control oligomerization state of the target protein and lipid composition of the bilayer. Special attention is paid to the opportunities afforded by nanodisc system for the detailed studies of the role of different lipid properties and protein-lipid interactions in the functional behavior of membrane proteins.
Assuntos
Bicamadas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Nanoestruturas/química , Nanotecnologia/métodos , Sequência de Aminoácidos , Animais , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação ProteicaRESUMO
PREMISE: Gravity is an important environmental factor that affects growth and development of plants. In response to changes in gravity, directional growth occurs along the major axes and lateral branches of both shoots and roots. The gravity persistent signal (gps) mutants of Arabidopsis thaliana were previously identified as having an altered response to gravity when reoriented relative to the gravity vector in the cold, with the gps1 mutant exhibiting a complete loss of tropic response under these conditions. METHODS: Thermal asymmetric interlaced (TAIL) PCR was used to identify the gene defective in gps1. Gene expression data, molecular modeling and computational substrate dockings, quantitative RT-PCR analyses, reporter gene fusions, and physiological analyses of knockout mutants were used to characterize the genes identified. RESULTS: Cloning of the gene defective in gps1 and genetic complementation revealed that GPS1 encodes CYP705A22, a cytochrome P450 monooxygenase (P450). CYP705A5, a closely related family member, was identified as expressed specifically in roots in response to gravistimulation, and a mutation affecting its expression resulted in a delayed gravity response, increased flavonol levels, and decreased basipetal auxin transport. Molecular modeling coupled with in silico substrate docking and diphenylboric acid 2-aminoethyl ester (DBPA) staining indicated that these P450s are involved in biosynthesis of flavonoids potentially involved in auxin transport. CONCLUSION: The characterization of two novel P450s (CYP705A22 and CYP705A5) and their role in the gravity response has offered new insights into the regulation of the genetic and physiological controls of plant gravitropism.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Gravitropismo/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Loci Gênicos/genética , Gravitropismo/efeitos dos fármacos , Mutação/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Fenótipo , Fosfatos/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
We describe cloning and characterization of three rice (Oryza sativa) NADPH-cytochrome P450 reductases (OsCPRs; E.C.1.6.2.4) that are potential donors to plant P450s, including tryptamine 5-hydroxylase (T5H) in serotonin synthesis and cinnamate 4-hydroxylase (C4H) in phenylpropanoid synthesis. All three OsCPR transcripts are induced to varying degrees by stresses. Co-expression of full-length OsCPR1, OsCPR2 and OsCPR3 with either T5H or C4H in E. coli indicated that the OsCPR2/T5H and OsCPR2/C4H constructs displayed the highest T5H and C4H catalytic activities. The N-terminal residues of OsCPR2 were required for peak electron transfer activity to P450 even though deletion mutants with short N-terminal deletions were capable of reducing cytochrome c.
Assuntos
Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação da Expressão Gênica , Oryza/enzimologia , Biotecnologia/métodos , Clonagem Molecular , Ácidos Cumáricos/química , Sistema Enzimático do Citocromo P-450/genética , Citocromos c/metabolismo , Escherichia coli/metabolismo , Deleção de Genes , Isoenzimas/genética , Isoenzimas/metabolismo , Oryza/genética , Propionatos , Proteínas Recombinantes/metabolismo , Serotonina/metabolismo , Transcinamato 4-Mono-Oxigenase/metabolismoRESUMO
Identification of the causal genes that control complex trait variation remains challenging, limiting our appreciation of the evolutionary processes that influence polymorphisms in nature. We cloned a quantitative trait locus that controls plant defensive chemistry, damage by insect herbivores, survival, and reproduction in the natural environments where this polymorphism evolved. These ecological effects are driven by duplications in the BCMA (branched-chain methionine allocation) loci controlling this variation and by two selectively favored amino acid changes in the glucosinolate-biosynthetic cytochrome P450 proteins that they encode. These changes cause a gain of novel enzyme function, modulated by allelic differences in catalytic rate and gene copy number. Ecological interactions in diverse environments likely contribute to the widespread polymorphism of this biochemical function.
Assuntos
Brassicaceae , Sistema Enzimático do Citocromo P-450/genética , Glucosinolatos/genética , Locos de Características Quantitativas , Característica Quantitativa Herdável , Seleção Genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Brassicaceae/genética , Brassicaceae/metabolismo , Brassicaceae/parasitologia , Dosagem de Genes , Interação Gene-Ambiente , Glucosinolatos/biossíntese , Herbivoria/fisiologia , Metionina/genética , Metionina/metabolismo , Dados de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Polimorfismo GenéticoRESUMO
Transcriptome profiling methods are rapidly changing the ways in which insect responses to the environment can be assessed. One article featured in this issue of Molecular Ecology utilizes global expression analysis to extend previous studies examining the basis of insecticide resistance in Helicoverpa species that are devastating crop pests worldwide. In this issue Tao et al. (2012) have used a wide spectrum of biochemical and molecular techniques to examine the extent to which naturally occurring plant compounds and one synthetic insecticide change transcriptome profiles of Helicoverpa armigera larvae (Fig. 1) to enhance their adaptation to plant toxins and insecticides in their diet and environment. With their data highlighting an array of P450 transcripts and a few esterase transcripts that are correlated with increased tolerance to the insecticide deltamethrin, these researchers provide the first comprehensive evaluation of cytochrome P450 monooxygenase (P450), glutathione S-transferase (GST) and esterase (Est) gene responses in a lepidopteran species repeatedly subjected to pyrethroid selection. Building on this information, these researchers venture into the realm of the biorational in showing that RNAi-mediated destruction of one prominent toxin-inducible P450 transcript can increase sensitivity to deltamethrin, thereby compromising larval growth in the presence of this insecticide. The fact that the resulting levels of deltamethrin tolerance in the RNAi-silenced larvae are not restored completely to the levels in larvae whose defences are not stimulated by consuming plant compounds provides further evidence that their adaptive strategies for survival in toxin-rich environments include many different P450, esterase and, possibly, other genes. Together, their data highlight the complexity of the defence systems evolving in lepidopterans and other insect pests.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Gossipol/farmacologia , Resistência a Inseticidas/genética , Mariposas/genética , AnimaisRESUMO
Gene duplication and divergence are overwhelmingly considered to be the primary mechanisms by which cytochrome P450 monooxygenases (P450s) have radiated into a large and diverse gene superfamily. To address how environmental stress drives the fixation and diversification of gene duplications, we have analyzed Cyp12d1 and Cyp12d3, a pair of duplicated genes found in the sequenced Drosophila genomes of the melanogaster group. The paralog Cyp12d3, which is not found in Drosophila melanogaster, is basal to the melanogaster group, after it split from the obscura group (ca. 50 mya), and has a significant signature of positive selection in two species (D. sechellia and D. ananassae). Examination of the Cyp12d1 region in D. melanogaster wildtype and isoline populations revealed variation both in copy number and sequence, including splice-site variations, which certainly alter gene function. Further investigations of several strains have identified three cases in which differences in the Cyp12d1 gene region are associated with the differences in transcript abundance and transcriptional responses to the environmental stresses that have not been seen for other detoxificative loci. Together, these data highlight the value of using both macro- and microevolutionary approaches in studying the duplication and divergence events associated with detoxification genes and lay important groundwork for future studies in the field of evolutionary toxicogenomics, which uses the principles of phylogenetic analysis to predict possible enzymatic functions.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila/enzimologia , Drosophila/genética , Evolução Molecular , Variação Genética , Toxicogenética , Aminoácidos/genética , Animais , Sequência de Bases , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Meio Ambiente , Dosagem de Genes/genética , Regulação Enzimológica da Expressão Gênica , Genes de Insetos/genética , Loci Gênicos/genética , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Estresse Fisiológico/genética , Fatores de TempoRESUMO
BACKGROUND: Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. CONCLUSIONS/SIGNIFICANCE: These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.
Assuntos
Abelhas/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Meio Ambiente , Ativação Transcricional/efeitos dos fármacos , Animais , Carboidratos/farmacologia , Dieta , Mel , Xenobióticos/antagonistas & inibidores , Xenobióticos/farmacologiaRESUMO
Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., (13)C-(13)C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (-0.75) commensurate to the control (-0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.