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
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
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
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
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
AIM: The purpose of this study was to determine gap areas where North Carolina should implement strategies to promote the Institute of Medicine's recommendation of an 80 percent bachelor of science workforce. BACKGROUND: The North Carolina Action Coalition sought information about the nursing BSN and higher degree workforce and about human resource policies/strategies that promote BSN and higher degree education. METHOD: An electronic survey was used to query 120 acute care hospital chief nursing officers over a four-year period and 100 public health chief nursing officers over a two-year period. RESULTS: A majority of acute care and a minority of public health institutions had policies promoting BSN education. Barriers included lack of tuition reimbursement, scheduling/staffing issues, lack of local universities, and a perceived lack of value for nurses. A minority of respondents reported an 80 percent BSN workforce. CONCLUSION: Strategies are needed statewide to support nursing academic progression.
Assuntos
Bacharelado em Enfermagem , Enfermeiros Administradores , Humanos , North Carolina , Inquéritos e Questionários , Estados UnidosRESUMO
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
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
Although Apis mellifera, the western honey bee, has long encountered pesticides when foraging in agricultural fields, for two decades it has encountered pesticides in-hive in the form of acaricides to control Varroa destructor, a devastating parasitic mite. The pyrethroid tau-fluvalinate and the organophosphate coumaphos have been used for Varroa control, with little knowledge of honey bee detoxification mechanisms. Cytochrome P450-mediated detoxification contributes to pyrethroid tolerance in many insects, but specific P450s responsible for pesticide detoxification in honey bees (indeed, in any hymenopteran pollinator) have not been defined. We expressed and assayed CYP3 clan midgut P450s and demonstrated that CYP9Q1, CYP9Q2, and CYP9Q3 metabolize tau-fluvalinate to a form suitable for further cleavage by the carboxylesterases that also contribute to tau-fluvalinate tolerance. These in vitro assays indicated that all of the three CYP9Q enzymes also detoxify coumaphos. Molecular models demonstrate that coumaphos and tau-fluvalinate fit into the same catalytic pocket, providing a possible explanation for the synergism observed between these two compounds. Induction of CYP9Q2 and CYP9Q3 transcripts by honey extracts suggested that diet-derived phytochemicals may be natural substrates and heterologous expression of CYP9Q3 confirmed activity against quercetin, a flavonoid ubiquitous in honey. Up-regulation by honey constituents suggests that diet may influence the ability of honey bees to detoxify pesticides. Quantitative RT-PCR assays demonstrated that tau-fluvalinate enhances CYP9Q3 transcripts, whereas the pyrethroid bifenthrin enhances CYP9Q1 and CYP9Q2 transcripts and represses CYP9Q3 transcripts. The independent regulation of these P450s can be useful for monitoring and differentiating between pesticide exposures in-hive and in agricultural fields.
Assuntos
Acaricidas/farmacocinética , Abelhas/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Insetos/metabolismo , Acaricidas/toxicidade , Sequência de Aminoácidos , Animais , Abelhas/parasitologia , Cumafos/farmacocinética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/classificação , Sistema Enzimático do Citocromo P-450/genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Parasita , Inativação Metabólica , Proteínas de Insetos/genética , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/farmacologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Nitrilas/química , Nitrilas/metabolismo , Nitrilas/farmacologia , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Piretrinas/química , Piretrinas/metabolismo , Piretrinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Varroidae/efeitos dos fármacos , Varroidae/fisiologiaRESUMO
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
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
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
Cytochrome P450 monooxygenases (P450s) are integral in defining the relationships between plants and insects. Secondary metabolites produced in plants for protection against insects and other organisms are synthesized via pathways that include P450s in many different families and subfamilies. Survival of insects in the presence of toxic secondary metabolites depends on their metabolism by more limited groups of P450s. Examples of functionally characterized plant and insect P450s known to be involved in these interactions are discussed in terms of their diversities, reactivities and regulators. These and future examples, which will be uncovered as the fields of plant biology and entomology converge on this interesting area, provide much insight into the array of plant metabolites that are mainline defenses against insects, the range of insect monooxygenases that inactivate these compounds and the evolutionary processes occurring as these organisms wage daily battles with one another. Molecular perspectives on these interactions will provide the scientific community with information critical for genetic manipulation of these organisms aimed at enhancing plant resistance to insects and eliminating insect resistance to natural plant toxins and synthetic insecticides.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Insetos/metabolismo , Insetos/enzimologia , Proteínas de Plantas/metabolismo , Plantas/enzimologia , Animais , Domínio Catalítico/genética , Sistema Enzimático do Citocromo P-450/genética , Evolução Molecular , Interações Hospedeiro-Parasita , Proteínas de Insetos/genética , Insetos/genética , Insetos/fisiologia , Proteínas de Plantas/genética , Plantas/genética , Plantas/parasitologiaRESUMO
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
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.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , Proteínas/química , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Aminoácidos/química , Bases de Dados de Proteínas , Modelos Moleculares , Conformação ProteicaRESUMO
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
An RN refresher program can be a valuable tool to mobilize nurses back into practice, but there are limited data regarding how much it increases students' confidence. Bandura's (1997) theory defines self-efficacy as a construct of one's belief in obtaining control over behavior and environment to achieve a goal. In this study, the validated Clinical Skills Self-Efficacy Scale (CSES) was used to measure the difference in a student's reported perception of self-efficacy after completing a refresher program. The CSES survey was distributed to two groups of RN students: 59 accelerated students who finished the course in 3 months and 57 traditional students who had 9 months to complete the course. The accelerated students had recent nursing experience, but were responding to the COVID-19 pandemic. A mixed model was used to analyze the CSES scores between the accelerated students and the traditional students. A pretest-posttest design was used for each item of the scale. Both groups of students had a statistically significant difference in their CSES scores from pretest to posttest (p < .05). [J Contin Educ Nurs. 2022;53(6):273-278.].
Assuntos
COVID-19 , Estudantes de Enfermagem , Competência Clínica , Humanos , Pandemias , AutoeficáciaRESUMO
The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-Å resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.
Assuntos
Actinomycetales/enzimologia , Antibacterianos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Teicoplanina/metabolismo , Actinomycetales/genética , Actinomycetales/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/genética , Dados de Sequência Molecular , Fenol/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
OBJECTIVE: To evaluate how stress at the larval stage alters adult mosquito performance and susceptibility to viral infection. METHODS: We used a model system consisting of Sindbis virus (SINV) and the yellow fever mosquito Aedes aegypti. Larvae were either reared under optimal conditions (control) or exposed to one of four types of stressors; suboptimal nutrients, starvation, elevated temperature, and a low dose of the insecticide malathion and adult females were fed SINV infectious blood meal. Differential expressions of stress, immune-specific and detoxification genes was measured in fourth instar larvae (HSP70, HSP83, cecropin, defensin, transferrin and CYP6Z6) and 3-day-old females (cecropin, defensin, transferrin) to identify plausible molecular mechanisms associated with mosquito response to stress. RESULTS: There were stress-specific variations in mosquito performance (survival, development time, female size), but all stressors had a consistent effect of significantly increasing susceptibility to viral infection and dissemination relative to the controls. Three genes were up-regulated in fourth instar larvae exposed to temperature stress (cecropin, defensin and CYP6Z6) compared to single genes in suboptimal nutrient (cecropin) and malathion (transferrin) stress treatments and down-regulation of all the six genes in starvation treatments. In adult samples, transferrin was up-regulated in all but starvation treatments while defensin was up-regulated in starvation and temperature stress treatments. CONCLUSIONS: Stress during larval development may cause alterations in adult mosquito phenotype and immunity that can increase their susceptibility to pathogens.