RESUMO
An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,ß-unsaturated compounds.
RESUMO
A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420 This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420 mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCE This study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobials in vitro and in vivoM. smegmatis strains unable to make or reduce F420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420 to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.
RESUMO
We have known for 40 years that soils can consume the trace amounts of molecular hydrogen (H2) found in the Earth's atmosphere.This process is predicted to be the most significant term in the global hydrogen cycle. However, the organisms and enzymes responsible for this process were only recently identified. Pure culture experiments demonstrated that several species of Actinobacteria, including streptomycetes and mycobacteria, can couple the oxidation of atmospheric H2 to the reduction of ambient O2. A combination of genetic, biochemical, and phenotypic studies suggest that these organisms primarily use this fuel source to sustain electron input into the respiratory chain during energy starvation. This process is mediated by a specialized enzyme, the group 5 [NiFe]-hydrogenase, which is unusual for its high affinity, oxygen insensitivity, and thermostability. Atmospheric hydrogen scavenging is a particularly dependable mode of energy generation, given both the ubiquity of the substrate and the stress tolerance of its catalyst. This minireview summarizes the recent progress in understanding how and why certain organisms scavenge atmospheric H2. In addition, it provides insight into the wider significance of hydrogen scavenging in global H2 cycling and soil microbial ecology.
Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/metabolismo , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Actinobacteria/enzimologia , Actinobacteria/genética , Microbiologia do Ar , Atmosfera/química , Proteínas de Bactérias/genética , Ecossistema , Hidrogenase/genéticaRESUMO
The metabolism of volatile signal molecules by odorant degrading enzymes (ODEs) is crucial to the ongoing sensitivity and specificity of chemoreception in various insects, and a few specific esterases, cytochrome P450s, glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs) have previously been implicated in this process. Significant progress has been made in characterizing ODEs in Lepidoptera but very little is known about them in Diptera, including in Drosophila melanogaster, a major insect model. We have therefore carried out a transcriptomic analysis of the antennae of D. melanogaster in order to identify candidate ODEs. Virgin male and female and mated female antennal transcriptomes were determined by RNAseq. As with the Lepidoptera, we found that many esterases, cytochrome P450 enzymes, GSTs and UGTs are expressed in D. melanogaster antennae. As olfactory genes generally show selective expression in the antennae, a comparison to previously published transcriptomes for other tissues has been performed, showing preferential expression in the antennae for one esterase, JHEdup, one cytochrome P450, CYP308a1, and one GST, GSTE4. These largely uncharacterized enzymes are now prime candidates for ODE functions. JHEdup was expressed heterologously and found to have high catalytic activity against a chemically diverse group of known ester odorants for this species. This is a finding consistent with an ODE although it might suggest a general role in clearing several odorants rather than a specific role in clearing a particular odorant. Our findings do not preclude the possibility of odorant degrading functions for other antennally expressed esterases, P450s, GSTs and UGTs but, if so, they suggest that these enzymes also have additional functions in other tissues.
Assuntos
Antenas de Artrópodes/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Enzimas/genética , Enzimas/metabolismo , Odorantes , Animais , Sequência de Bases , Feminino , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Reprodução/fisiologia , Fatores Sexuais , TranscriptomaRESUMO
Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.
Assuntos
Apoenzimas/biossíntese , Arthrobacter/enzimologia , Hidrolases/biossíntese , Apoenzimas/química , Apoenzimas/genética , Simulação por Computador , Cristalografia por Raios X , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Herbicidas/metabolismo , Hidrolases/química , Hidrolases/genética , Hidrólise , Cinética , Modelos Moleculares , Proteínas Mutantes/biossíntese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade , Triazinas/metabolismoRESUMO
Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.
Assuntos
Bactérias/química , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Colorimetria/métodos , Hexaclorocicloexano/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Hidrolases/metabolismo , Liases/metabolismo , Sequência de Aminoácidos , Bactérias/isolamento & purificação , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Halogenação , Hexaclorocicloexano/química , Hidrolases/química , Hidrolases/genética , Isomerismo , Cinética , Liases/química , Liases/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.
Assuntos
Esterases/genética , Esterases/metabolismo , Lepidópteros/enzimologia , Mariposas/enzimologia , Mutação/genética , Organofosfatos/metabolismo , Piretrinas/metabolismo , Animais , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Hidrólise , Inseticidas , Lepidópteros/genética , Lepidópteros/metabolismo , Leucina/genética , Leucina/metabolismo , Mariposas/genética , Mariposas/metabolismoRESUMO
Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.
RESUMO
Pandoraea sp. strain SD6-2 is a δ-hexachlorocyclohexane-degrading bacterial strain isolated from lindane-contaminated soil in Queensland, Australia. The genome of SD6-2 was sequenced to investigate its ability to degrade δ-hexachlorocyclohexane. Here we report the annotated genome sequence of this strain.
RESUMO
Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.
Assuntos
Esterases/genética , Mariposas/enzimologia , Animais , Arildialquilfosfatase/metabolismo , DNA Complementar , Esterases/metabolismo , Etiquetas de Sequências Expressas , Glicosilfosfatidilinositóis/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Eletroforese em Gel de Poliacrilamida NativaRESUMO
Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of organophosphate (OP) insecticides. Despite the importance of OPs to agriculture and the spread of insect-borne diseases, the molecular basis for the ability of α-carboxylesterases to confer OP resistance to insects is poorly understood. In this work, we used laboratory evolution to increase the thermal stability of LcαE7, allowing its overexpression in Escherichia coli and structure determination. The crystal structure reveals a canonical α/ß-hydrolase fold that is very similar to the primary target of OPs (acetylcholinesterase) and a unique N-terminal α-helix that serves as a membrane anchor. Soaking of LcαE7 crystals in OPs led to the capture of a crystallographic snapshot of LcαE7 in its phosphorylated state, which allowed comparison with acetylcholinesterase and rationalization of its ability to protect insects against the effects of OPs. Finally, inspection of the active site of LcαE7 reveals an asymmetric and hydrophobic substrate binding cavity that is well-suited to fatty acid methyl esters, which are hydrolyzed by the enzyme with specificity constants (â¼10(6) M(-1) s(-1)) indicative of a natural substrate.
Assuntos
Carboxilesterase/química , Carboxilesterase/metabolismo , Dípteros/efeitos dos fármacos , Dípteros/enzimologia , Resistência a Medicamentos/fisiologia , Inseticidas/química , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Austrália , Carboxilesterase/genética , Domínio Catalítico/fisiologia , Cristalografia por Raios X , Genes de Insetos/fisiologia , Fosforilação/fisiologia , Estrutura Secundária de Proteína/fisiologia , Ovinos , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/prevenção & controle , Especificidade por SubstratoRESUMO
Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L⻹ of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC50 values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Resistência a Inseticidas , Inseticidas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/isolamento & purificação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Inseticidas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
The widely accepted paradigm for the development of insecticide resistance in field populations of insects is of selection for one or a very few genes of major effect. Limited genetic mapping data for organophosphate and pyrethroid resistance in heliothine and spodopteran pests generally agrees with this paradigm. However, other biochemical and transcriptomic data suggest a more complex set of changes in multiple P450 and esterase gene/enzyme systems in resistant strains of these species. We discuss possible explanations for this paradox, including the likely embedding of these genes in regulatory cascades and emerging evidence for their arrangement in large clusters of closely related genes. We conclude that there could indeed be an unusually large number of genetic options for evolving resistance in these species.
Assuntos
Evolução Biológica , Proteínas de Insetos/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/genética , Spodoptera/genética , Animais , Proteínas de Insetos/metabolismo , Mariposas/efeitos dos fármacos , Mariposas/enzimologia , Spodoptera/efeitos dos fármacos , Spodoptera/enzimologiaRESUMO
A 6-chloronicotinic acid mineralizing bacterium was isolated from enrichment cultures originating from imidacloprid-contaminated soil samples. This Bradyrhizobiaceae, designated strain SG-6C, hydrolytically dechlorinated 6-chloronicotinic acid to 6-hydroxynicotinic acid, which was then further metabolised via the nicotinic acid pathway. This metabolic pathway was confirmed by growth and resting cell assays using HPLC and LC-MS studies. A candidate for the gene encoding the initial dechlorination step, named cch2 (for 6-chloronicotinic acid chlorohydrolase), was identified using genome sequencing and its function was confirmed using resting cell assays on E. coli heterologously expressing this gene. The 464 amino acid enzyme was found to be a member of the metal dependent hydrolase superfamily with similarities to the TRZ/ATZ family of chlorohydrolases. We also provide evidence that cch2 was mobilized into this bacterium by an Integrative and Conjugative Element (ICE) that feeds 6-hydroxynicotinic acid into the existing nicotinic acid mineralization pathway.
Assuntos
Bradyrhizobiaceae/enzimologia , Bradyrhizobiaceae/isolamento & purificação , Hidrolases/genética , Minerais/metabolismo , Ácidos Nicotínicos/metabolismo , Biodegradação Ambiental , Bradyrhizobiaceae/genética , Bradyrhizobiaceae/crescimento & desenvolvimento , Clonagem Molecular , Conjugação Genética/genética , Genes Bacterianos/genética , Hidrolases/metabolismo , Ácidos Nicotínicos/químicaRESUMO
Atrazine chlorohydrolase (AtzA) and its close relative melamine deaminase (TriA) differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.
Assuntos
Aminoidrolases/genética , Epistasia Genética , Evolução Molecular , Hidrolases/genética , Pseudomonas/enzimologia , Substituição de Aminoácidos/genética , Aminoácidos/metabolismo , Aminoidrolases/química , Aminoidrolases/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Hidrolases/química , Hidrolases/metabolismo , Cinética , Modelos Moleculares , Temperatura de TransiçãoRESUMO
Two classes of F(420)-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F(420) is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F(420)H(2) to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,ß-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F(420) to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products.
Assuntos
Aflatoxinas/metabolismo , Furocumarinas/química , Regulação da Expressão Gênica , Mycobacterium smegmatis/metabolismo , Oxirredutases/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Cumarínicos/química , Flavinas/química , Hidrólise , Quelina/química , Espectrometria de Massas/métodos , Metoxaleno/química , Filogenia , Especificidade da Espécie , Xenobióticos/farmacologiaRESUMO
Esterases have been implicated in metabolic resistance to synthetic pyrethroids in several insect species but little is yet known of the molecular basis for these effects. In this work modern directed evolution technology was used to test to what extent it is possible to genetically enhance the pyrethroid hydrolytic activity of the E3 carboxylesterase from the blowfly Lucilia cuprina. High throughput screening of a random mutant library with individual stereoisomers of fluorogenic analogues of two type II pyrethroids identified 17 promising variants that were then also tested with the commercial pyrethroid deltamethrin. Between them, these variants displayed significantly improved activities for all the substrates tested. Amino acid substitutions at ten different residues were clearly implicated in the improvements, although most only enhanced activity for a subset of the stereoisomers. Several new combinations of the most promising amino acid substitutions were then made, and negative epistatic effects were found in most of the combinations, but significant improvements were also found in a minority of them. The best mutant recovered contained three amino acid changes and hydrolysed deltamethrin at more than 100 times the rate of wild-type E3. Structural analysis shows that nine of the ten mutated residues improving pyrethroid or analogue activities cluster in putative substrate binding pockets in the active site, with the three mutations of largest effect all increasing the volume of the acyl pocket.
Assuntos
Carboxilesterase/genética , Dípteros/enzimologia , Evolução Molecular , Inseticidas , Nitrilas , Piretrinas , Substituição de Aminoácidos , Animais , Carboxilesterase/metabolismo , Dípteros/genética , Escherichia coli , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Mutação , Estrutura Terciária de Proteína , Recombinação Genética , EstereoisomerismoRESUMO
BACKGROUND: Here we report specific activities of all seven naturally occurring LinA variants towards three different isomers, α, γ and δ, of a priority persistent pollutant, hexachlorocyclohexane (HCH). Sequence-structure-function differences contributing to the differences in their stereospecificity for α-, γ-, and δ-HCH and enantiospecificity for (+)- and (-)-α -HCH are also discussed. METHODOLOGY/PRINCIPAL FINDINGS: Enzyme kinetic studies were performed with purified LinA variants. Models of LinA2(B90A) A110T, A111C, A110T/A111C and LinA1(B90A) were constructed using the FoldX computer algorithm. Turnover rates (min(-1)) showed that the LinAs exhibited differential substrate affinity amongst the four HCH isomers tested. α-HCH was found to be the most preferred substrate by all LinA's, followed by the γ and then δ isomer. CONCLUSIONS/SIGNIFICANCE: The kinetic observations suggest that LinA-γ1-7 is the best variant for developing an enzyme-based bioremediation technology for HCH. The majority of the sequence variation in the various linA genes that have been isolated is not neutral, but alters the enantio- and stereoselectivity of the encoded proteins.
Assuntos
Anticonvulsivantes/metabolismo , Proteínas de Bactérias/metabolismo , Variação Genética/genética , Hexaclorocicloexano/metabolismo , Liases/metabolismo , Anticonvulsivantes/química , Proteínas de Bactérias/genética , Biodegradação Ambiental , Escherichia coli/enzimologia , Hexaclorocicloexano/química , Cinética , Liases/genética , Modelos Químicos , EstereoisomerismoRESUMO
Strain SG-6C (DSM 23264, CCM 7827) is a chemolithoautotrophic bacterium of the family Bradyrhizobiaceae. It can also grow heterotrophically under appropriate environmental conditions. Here we report the annotated genome sequence of this strain in a single 4.3-Mb circular scaffold.
Assuntos
Bradyrhizobiaceae/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Genoma Bacteriano , Análise de Sequência de DNA , Austrália , Bradyrhizobiaceae/isolamento & purificação , Bradyrhizobiaceae/fisiologia , Crescimento Quimioautotrófico , Processos Heterotróficos , Dados de Sequência Molecular , Microbiologia do SoloRESUMO
Esters are an important component of apple (Malus×domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, 'Royal Gala', that contain all the conserved features associated with CXE members of the α/ß hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of 'Royal Gala' (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K(m)) but decreasing turnover (k(cat)) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.