RESUMEN
Adenylate kinases (AKs) have evolved AMP-binding and lid domains that are encoded as continuous polypeptides embedded at different locations within the discontinuous polypeptide encoding the core domain. A prior study showed that AK homologues of different stabilities consistently retain cellular activity following circular permutation that splits a region with high energetic frustration within the AMP-binding domain into discontinuous fragments. Herein, we show that mesophilic and thermophilic AKs having this topological restructuring retain activity and substrate-binding characteristics of the parental AK. While permutation decreased the activity of both AK homologues at physiological temperatures, the catalytic activity of the thermophilic AK increased upon permutation when assayed >30 °C below the melting temperature of the native AK. The thermostabilities of the permuted AKs were uniformly lower than those of native AKs, and they exhibited multiphasic unfolding transitions, unlike the native AKs, which presented cooperative thermal unfolding. In addition, proteolytic digestion revealed that permutation destabilized each AK in differing manners, and mass spectrometry suggested that the new termini within the AMP-binding domain were responsible for the increased proteolysis sensitivity. These findings illustrate how changes in contact order can be used to tune enzyme activity and alter folding dynamics in multidomain enzymes.
Asunto(s)
Adenilato Quinasa , Péptidos , Adenilato Quinasa/química , Secuencia de Aminoácidos , TemperaturaRESUMEN
In early 2022, a cluster of monkeypox virus (MPXV) infection (mpox) cases were identified within the UK with no prior travel history to MPXV-endemic regions. Subsequently, case numbers exceeding 80,000 were reported worldwide, primarily affecting gay, bisexual, and other men who have sex with men (GBMSM). Public health agencies worldwide have offered the IMVANEX Smallpox vaccination to these individuals at high-risk to provide protection and limit the spread of MPXV. We have developed a comprehensive array of ELISAs to study poxvirus-induced antibodies, utilising 24 MPXV and 3 Vaccinia virus (VACV) recombinant antigens. Panels of serum samples from individuals with differing Smallpox-vaccine doses and those with prior MPXV infection were tested on these assays, where we observed that one dose of Smallpox vaccination induces a low number of antibodies to a limited number of MPXV antigens but increasing with further vaccination doses. MPXV infection induced similar antibody responses to diverse poxvirus antigens observed in Smallpox-vaccinated individuals. We identify MPXV A27 as a serological marker of MPXV-infection, whilst MPXV M1 (VACV L1) is likely IMVANEX-specific. Here, we demonstrate analogous humoral antigen recognition between both MPXV-infected or Smallpox-vaccinated individuals, with binding to diverse yet core set of poxvirus antigens, providing opportunities for future vaccine (e.g., mRNA) and therapeutic (e.g., mAbs) design.
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Minorías Sexuales y de Género , Vacuna contra Viruela , Viruela , Masculino , Humanos , Monkeypox virus/genética , Viruela/prevención & control , Inmunidad Humoral , Homosexualidad MasculinaRESUMEN
The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a γ-keto-α,ß-unsaturated aldehyde species which could be trapped inâ vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds.
Asunto(s)
Ácido Benzoico , Sistema Enzimático del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-Reducción , Óxidos , TiofenosRESUMEN
Together with bacteria and filamentous fungi, yeasts actively take part in the global carbon cycle. Over 100 yeast species have been shown to grow on the major plant polysaccharide xylan, which requires an arsenal of carbohydrate active enzymes. However, which enzymatic strategies yeasts use to deconstruct xylan and what specific biological roles they play in its conversion remain unclear. In fact, genome analyses reveal that many xylan-metabolizing yeasts lack expected xylanolytic enzymes. Guided by bioinformatics, we have here selected three xylan-metabolizing ascomycetous yeasts for in-depth characterization of growth behavior and xylanolytic enzymes. The savanna soil yeast Blastobotrys mokoenaii displays superior growth on xylan thanks to an efficient secreted glycoside hydrolase family 11 (GH11) xylanase; solving its crystal structure revealed a high similarity to xylanases from filamentous fungi. The termite gut-associated Scheffersomyces lignosus, in contrast grows more slowly, and its xylanase activity was found to be mainly cell surface-associated. The wood-isolated Wickerhamomyces canadensis, surprisingly, could not utilize xylan as the sole carbon source without the addition of xylooligosaccharides or exogenous xylanases or even co-culturing with B. mokoenaii, suggesting that W. canadensis relies on initial xylan hydrolysis by neighboring cells. Furthermore, our characterization of a novel W. canadensis GH5 subfamily 49 (GH5_49) xylanase represents the first demonstrated activity in this subfamily. Our collective results provide new information on the variable xylanolytic systems evolved by yeasts and their potential roles in natural carbohydrate conversion. IMPORTANCE Microbes that take part in the degradation of the polysaccharide xylan, the major hemicellulose component in plant biomass, are equipped with specialized enzyme machineries to hydrolyze the polymer into monosaccharides for further metabolism. However, despite being found in virtually every habitat, little is known of how yeasts break down and metabolize xylan and what biological role they may play in its turnover in nature. Here, we have explored the enzymatic xylan deconstruction strategies of three underexplored yeasts from diverse environments, Blastobotrys mokoenaii from soil, Scheffersomyces lignosus from insect guts, and Wickerhamomyces canadensis from trees, and we show that each species has a distinct behavior regarding xylan conversion. These findings may be of high relevance for future design and development of microbial cell factories and biorefineries utilizing renewable plant biomass.
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Polisacáridos , Xilanos , Xilanos/metabolismo , Polisacáridos/metabolismo , Levaduras , Hongos/metabolismoRESUMEN
Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases which catalyze the oxidation of aliphatic and aromatic C-H bonds and other reactions. The oxidation of halogens by cytochrome P450 enzymes has also been reported. Here we use CYP199A4, from the bacterium Rhodopseudomonas palustris strain HaA2, with a range of para-substituted benzoic acid ligands, which contain halogens, to assess if this enzyme can oxidize these species or if the presence of these electronegative atoms can alter the outcome of P450-catalyzed reactions. Despite binding to the enzyme, there was no detectable oxidation of any of the 4-halobenzoic acids. CYP199A4 was, however, able to efficiently catalyze the oxidation of both 4-chloromethyl- and 4-bromomethyl-benzoic acid to 4-formylbenzoic acid via hydroxylation of the αcarbon. The 4-chloromethyl substrate bound in the enzyme active site in a similar manner to 4-ethylbenzoic acid. This places the benzylic αcarbon hydrogens in an unfavorable position for abstraction indicating a degree of substrate mobility must be possible within the active site. CYP199A4 catalyzed oxidations of 4-(2'-haloethyl)benzoic acids yielding α-hydroxylation and desaturation metabolites. The α-hydroxylation product was the major metabolite. The desaturation pathway is significantly disfavored compared to 4-ethylbenzoic acid. This may be due to the electron-withdrawing halogen atom or a different positioning of the substrate within the active site. The latter was demonstrated by the X-ray crystal structures of CYP199A4 with these substrates. Overall, the presence of a halogen atom positioned close to the heme iron can alter the binding orientation and outcomes of enzyme-catalyzed oxidation.
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Ácido Benzoico , Sistema Enzimático del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-Reducción , Catálisis , Hemo/química , HidroxilaciónRESUMEN
The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cß carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cß position (41%). The oxidation of 4-n-propylbenzoic acid by the F298â V mutant of CYP199A4 occurred with no hydroxylation at Cß and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298â V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
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Sistema Enzimático del Citocromo P-450 , Hemo , Especificidad por Sustrato , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/química , Catálisis , Alquenos , CarbonoRESUMEN
Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H2 O2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H2 O2 complex which would be more abundant in the peroxide-driven reactions.
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Compuestos Férricos , Oxidantes , Oxidantes/química , Sistema Enzimático del Citocromo P-450/metabolismo , Hemo/química , CatálisisRESUMEN
The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid; however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.
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Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450 , Hidroxilación , Especificidad por Sustrato , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ingeniería de Proteínas , Hemo/química , Oxidación-Reducción , Benzoatos/químicaRESUMEN
The Omicron, or Pango lineage B.1.1.529, variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries multiple spike mutations with high transmissibility and partial neutralizing antibody (nAb) escape. Vaccinated individuals show protection against severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple BioNTech BNT162b2 messenger RNA-vaccinated health care workers (HCWs) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple-vaccinated individuals, but the magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCWs who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529.
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Linfocitos B , Vacuna BNT162 , COVID-19 , Inmunización Secundaria , SARS-CoV-2 , Linfocitos T , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Vacuna BNT162/inmunología , Vacuna BNT162/uso terapéutico , COVID-19/inmunología , COVID-19/prevención & control , Reacciones Cruzadas , Humanos , Ratones , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunologíaRESUMEN
Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity.
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Tratamiento Farmacológico de COVID-19 , COVID-19 , Animales , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales , COVID-19/terapia , Análisis Costo-Beneficio , Inmunización Pasiva , SARS-CoV-2 , Ovinos , Glicoproteína de la Espiga del Coronavirus , Sueroterapia para COVID-19RESUMEN
Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small.
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COVID-19 , Ciervos , Animales , Animales Salvajes , Anticuerpos Antivirales , COVID-19/epidemiología , COVID-19/veterinaria , Prueba de COVID-19/veterinaria , Humanos , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.
Asunto(s)
Hidrolasas de Éster Carboxílico , Clostridium butyricum , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Clostridium butyricum/enzimología , Estructura Terciaria de Proteína , Especificidad por Sustrato , Taninos/químicaRESUMEN
The cytochrome P450 (CYP) superfamily of heme monooxygenases is involved in a range of important chemical biotransformations across nature. Azole-containing molecules have been developed as drugs that bind to the heme center of these enzymes, inhibiting their function. The optical spectrum of CYP enzymes after the addition of these inhibitors is used to assess how the molecules bind. Here we use the bacterial CYP199A4 enzyme, from Rhodopseudomonas palustris HaA2, to compare how imidazolyl and triazolyl inhibitors bind to ferric and ferrous heme. 4-(Imidazol-1-yl)benzoic acid induced a red shift in the Soret wavelength (424 nm) in the ferric enzyme along with an increase and a decrease in the intensities of the δ and α bands, respectively. 4-(1H-1,2,4-Triazol-1-yl)benzoic acid binds to CYP199A4 with a 10-fold lower affinity and induces a smaller red shift in the Soret band. The crystal structures of CYP199A4 with these two inhibitors confirmed that these differences in the optical spectra were due to coordination of the imidazolyl ligand to the ferric Fe, but the triazolyl inhibitor interacts with, rather than displaces, the ferric aqua ligand. Additional water molecules were present in the active site of 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound CYP199A4. The space required to accommodate these additional water molecules in the active site necessitates changes in the position of the hydrophobic phenylalanine 298 residue. Upon reduction of the heme, the imidazole-based inhibitor Fe-N ligation was not retained. A 5-coordinate heme was also the predominant species in 4-(1H-1,2,4-triazol-1-yl)benzoic acid-bound ferrous CYP199A4, but there was an obvious shoulder at 447 nm indicative of some degree of Fe-N coordination. Rather than inhibit CYP199A4, 4-(imidazol-1-yl)benzoic acid was a substrate and was oxidized to generate a metabolite derived from ring opening of the imidazolyl ring: 4-[[2-(formylamino)acetyl]amino]benzoic acid.
Asunto(s)
Sistema Enzimático del Citocromo P-450RESUMEN
The serine 244 to aspartate (S244D) variant of the cytochrome P450 enzyme CYP199A4 was used to expand its substrate range beyond benzoic acids. Substrates, in which the carboxylate group of the benzoic acid moiety is replaced were oxidised with high activity by the S244D mutant (product formation rates >60â nmol.(nmol-CYP)-1 .min-1 ) and with total turnover numbers of up to 20,000. Ethyl α-hydroxylation was more rapid than methyl oxidation, styrene epoxidation and S-oxidation. The S244D mutant catalysed the ethyl hydroxylation, epoxidation and sulfoxidation reactions with an excess of one stereoisomer (in some instances up to >98 %). The crystal structure of 4-methoxybenzoic acid-bound CYP199A4 S244D showed that the active site architecture and the substrate orientation were similar to that of the WT enzyme. Overall, this work demonstrates that CYP199A4 can catalyse the stereoselective hydroxylation, epoxidation or sulfoxidation of substituted benzene substrates under mild conditions resulting in more sustainable transformations using this heme monooxygenase enzyme.
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Benceno , Sistema Enzimático del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxilación , Oxidación-Reducción , Especificidad por SustratoRESUMEN
We investigated geographic variation in the semiochemistry of major disturbance agents of western North American pine forests, Dendroctonus brevicomis Le Conte and Dendroctonus barberi Hopkins (Coleoptera: Curculionidae: Scolytinae), species separated by the Great Basin in the USA that until recently were synonymous. At 15 sites in the western USA and northern Mexico, beetle populations were examined to determine (1) pheromone production by solitary, mining females, (2) male electroantennogram amplitudes in response to known semiochemicals for the genus, or (3) relative attractiveness of two female-produced pheromone components (endo- and exo-brevicomin) and two host odors (alpha-pinene and myrcene) to beetles in the field. Compared to female beetles collected east of the Great Basin (D. barberi), western females (D. brevicomis) produced a consistently higher proportion of, and male antenna were correspondingly more sensitive to, the exo- than the endo-isomer of brevicomin. With the exception of one sampling location (where no preference was observed), beetles west of the Great Basin were more attracted to exo- than endo- brevicomin trap lures, whereas eastern beetles displayed the reverse preference. In contrast, there was not a consistent difference between these populations regarding relative attraction or olfactory response to myrcene or alpha-pinene, although some geographic variability was evident. These data show that the semiochemical systems of D. brevicomis and D. barberi have diverged and corroborate genetic and morphological evidence that they are distinct, allopatric species.
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Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Escarabajos/química , Especiación Genética , Feromonas/química , Monoterpenos Acíclicos/metabolismo , Alquenos/metabolismo , Animales , Conducta Animal , Monoterpenos Bicíclicos/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/análisis , Escarabajos/fisiología , Femenino , Interacciones Huésped-Parásitos , Masculino , Feromonas/fisiología , Filogeografía , Pinus ponderosa/metabolismo , Pinus ponderosa/parasitología , Especificidad de la EspecieRESUMEN
The invasive ambrosia beetle polyphagous shot hole borer is one member of the cryptic species, Euwallacea nr. fornicatus (Eichhoff), and poses a great ecological and environmental threat to ornamental and native hardwood trees and agriculturally important tree crops in southern California, United States. We monitored the emergence of polyphagous shot hole borer adults from chipped and unchipped cut logs of infested boxelder, Acer negundo L. (Sapindales: Sapindaceae), California sycamore, Platanus racemosa Nutt. (Proteales: Platanaceae), coast live oak, Quercus agrifolia Née (Fagales: Fagaceae), and red willow, Salix laevigata Bebb (Malpighiales: Salicaceae), every week for 4 to 5 mo. No polyphagous shot hole borer adults emerged from chipped or unchipped coast live oak logs, suggesting this species is not a preferred reproductive host. However, following chipping, a small number of polyphagous shot hole borer adults emerged from boxelder (up to 7 wk) and from California sycamore and red willow (both up to 9 wk). A significantly greater number of polyphagous shot hole borer adults emerged from unchipped logs of boxelder (up to 14 wk) and California sycamore and red willow (both up to 5 mo). Chipping of boxelder, California sycamore, and red willow by using common commercially available chippers reduced polyphagous shot hole borer emergence by over 97%. Emerged polyphagous shot hole borer adults were strongly female-biased, regardless of host. Chipping treatments were highly effective, but for the complete elimination of polyphagous shot hole borer from woody material, other sanitation measures such as solarization in conjunction with chipping are recommended. Environmental and ecological impact of polyphagous shot hole borer is reviewed and discussed.
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Escarabajos , Gorgojos , Animales , Femenino , Reproducción , Saneamiento , ÁrbolesRESUMEN
Wildland fires have a multitude of ecological effects in forests, woodlands, and savannas across the globe. A major focus of past research has been on tree mortality from fire, as trees provide a vast range of biological services. We assembled a database of individual-tree records from prescribed fires and wildfires in the United States. The Fire and Tree Mortality (FTM) database includes records from 164,293 individual trees with records of fire injury (crown scorch, bole char, etc.), tree diameter, and either mortality or top-kill up to ten years post-fire. Data span 142 species and 62 genera, from 409 fires occurring from 1981-2016. Additional variables such as insect attack are included when available. The FTM database can be used to evaluate individual fire-caused mortality models for pre-fire planning and post-fire decision support, to develop improved models, and to explore general patterns of individual fire-induced tree death. The database can also be used to identify knowledge gaps that could be addressed in future research.
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Incendios , Agricultura Forestal , Bosques , Árboles , Bases de Datos como Asunto , Estados UnidosRESUMEN
Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, trees and stands can be protected from Douglas-fir beetle, Dendroctonus pseudotsugae Hopkins (DFB)-caused mortality by application of synthetic formulations of the beetle's antiaggregation pheromone, 3-methylcyclohex-2-en-1-one (MCH). A biodegradable formulation of MCH, SPLAT MCH, was developed and evaluated for protecting individual Douglas-fir trees and small stands from colonization and mortality by DFB. In an individual-tree experiment in Idaho, both MCH bubble capsules and SPLAT MCH significantly reduced the proportion of treated trees colonized and killed by DFB compared to untreated controls. SPLAT MCH was as effective as MCH bubble capsules for protecting individual trees. Both MCH bubble capsules and SPLAT MCH significantly reduced the proportion of trees colonized and killed by DFB within 0.04-ha circular plots surrounding each treated tree compared to untreated controls. In 0.41 ha stands in New Mexico, both MCH bubble capsules and SPLAT MCH significantly reduced the proportion of trees colonized and killed by DFB compared to untreated controls, again with no differences observed between MCH treatments. In a similar stand level trial in Idaho, neither MCH treatment significantly reduced the proportion of trees colonized by DFB, and only MCH bubble capsules significantly reduced levels of tree mortality compared to untreated controls, but no significant difference was observed between SPLAT MCH and MCH bubble capsules. Overall, the results indicate that SPLAT MCH is as effective as MCH bubble capsules for protecting individual trees and small stands of Douglas-fir from DFB-caused mortality.
Asunto(s)
Escarabajos , Pseudotsuga , Gorgojos , Animales , Ciclohexanos , Idaho , New Mexico , ÁrbolesRESUMEN
The cytochrome P450 heme monooxygenases commonly use an acid-alcohol pair of residues, within the I-helix, to activate iron-bound dioxygen. This work aims to clarify conflicting reports on the importance of the alcohol functionality in this process. Mutants of the P450, CYP199A4 (CYP199A4D251N and CYP199A4T252A), were prepared, characterised and their crystal structures were solved. The acid residue of CYP199A4 is not part of a salt bridge network, a key feature of paradigmatic model system P450cam. Instead, there is a direct proton delivery network, via a chain of water molecules, extending to the surface. Nevertheless, CYP199A4D251N dramatically reduced the activity of the enzyme consistent with a role in proton delivery. CYP199A4T252A decreased the coupling efficiency of the enzyme with a concomitant increase in the hydrogen peroxide uncoupling pathway. However, the effect of this mutation was much less pronounced than reported with P450cam. Its crystal structures revealed fewer changes at the I-helix, compared to the P450cam system. The structural changes observed within the I-helix of P450cam during oxygen activation do not seem to be required in this P450. These differences are due to the presence of a second threonine residue at position 253, which is absent in P450cam. This threonine forms part of the hydrogen bonding network, resulting in subtle structural changes and is also present across the majority of the P450 superfamily. Overall, the results suggest that while the acid-alcohol pair is important for dioxygen activation this process and the method of proton delivery can differ across P450s.Graphic abstract.
Asunto(s)
Alcoholes/química , Benzoatos/química , Sistema Enzimático del Citocromo P-450/química , Oxígeno/química , Alcoholes/metabolismo , Benzoatos/metabolismo , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Oxígeno/metabolismoRESUMEN
The cytochrome P450 superfamily of heme monooxygenases catalyzes important chemical reactions across nature. The changes in the optical spectra of these enzymes, induced by the addition of substrates or inhibitors, are critical for assessing how these molecules bind to the P450, enhancing or inhibiting the catalytic cycle. Here we use the bacterial CYP199A4 enzyme (Uniprot entry Q2IUO2), from Rhodopseudomonas palustris HaA2, and a range of substituted benzoic acids to investigate different binding modes. 4-Methoxybenzoic acid elicits an archetypal type I spectral response due to a ≥95% switch from the low- to high-spin state with concomitant dissociation of the sixth aqua ligand. 4-(Pyridin-3-yl)- and 4-(pyridin-2-yl)benzoic acid induced different type II ultraviolet-visible (UV-vis) spectral responses in CYP199A4. The former induced a greater red shift in the Soret wavelength (424 nm vs 422 nm) along with a larger overall absorbance change and other differences in the α-, ß-, and δ-bands. There were also variations in the ferrous UV-vis spectra of these two substrate-bound forms with a spectrum indicative of Fe-N bond formation with 4-(pyridin-3-yl)benzoic acid. The crystal structures of CYP199A4, with the pyridinyl compounds bound, revealed that while the nitrogen of 4-(pyridin-3-yl)benzoic acid is coordinated to the heme, with 4-(pyridin-2-yl)benzoic acid an aqua ligand remains. Continuous wave and pulse electron paramagnetic resonance data in frozen solution revealed that the substrates are bound in the active site in a form consistent with the crystal structures. The redox potential of each CYP199A4-substrate combination was measured, allowing correlation among binding modes, spectroscopic properties, and the observed biochemical activity.