RESUMO
Organophosphorus (OP) pesticides are still widely applied but pose a severe toxicological threat if misused. For in vivo detoxification, the application of hydrolytic enzymes potentially offers a promising treatment. A well-studied example is the phosphotriesterase of Brevundimonas diminuta (BdPTE). Whereas wild-type BdPTE can hydrolyse pesticides like paraoxon, chlorpyrifos-oxon and mevinphos with high catalytic efficiencies, kcat/KM >2 × 107 M-1 min-1, degradation of malaoxon is unsatisfactory (kcat/KM ≈ 1 × 104 M-1 min-1). Here, we report the rational engineering of BdPTE mutants with improved properties and their efficient production in Escherichia coli. As result, the mutant BdPTE(VRNVVLARY) exhibits 37-fold faster malaoxon hydrolysis (kcat/KM = 4.6 × 105 M-1 min-1), together with enhanced expression yield, improved thermal stability and reduced susceptibility to oxidation. Therefore, this BdPTE mutant constitutes a powerful candidate to develop a biocatalytic antidote for the detoxification of this common pesticide metabolite as well as related OP compounds.
Assuntos
Praguicidas , Hidrolases de Triester Fosfórico , Praguicidas/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Malation , Compostos Organofosforados/metabolismoRESUMO
The problem of biofilm formation is a serious concern under various pathological conditions such as extensive burns, wounds in diabetic patients, bedsores, cystic fibrosis, nosocomial infections from implantable medical devices such as catheters, valves, etc. Environmental diffusion of biofilm (in pools, wet floors, industrial food plants) that could represent a reservoir of antibiotic resistant bacteria constitues an additional issue. In this work is described a lactonase from Rhodococcus erythropolis, a phosphotriesterase-like lactonase (PLL) enzyme, which has already been studied in the past and can be used for containment of biofilm formation. The protein is 28% and 40% identical with respect to the Pseudomonas diminuta PTE and the thermostable Saccharolobus solfataricus SsoPox respectively. The protein was obtained starting from a synthetic His-tagged gene, expressed in E. coli, purified and further characterized. New properties, not previously known or deducible from its sequence, have been highlighted. These properties are: the enzyme is thermophilic and thermostable even though it originates from a mesophilic bacterium; the enzyme has a long (months) shelf life at 4 °C; the enzyme is not only stable to low concentrations of the oxidant H2O2 but even activated by it at high concentrations; the enzyme proved to be a proficient quorum quenching enzyme, able to hydrolase acyl-homoserine lactones 3oxoC12-HSL and C4-HSL, and can inhibit up to 60% the formation of Pseudomonas aeruginosa (PAO1) biofilm. These different properties make the lactonase useful to fight resistant bacteria that induce inflammatory and infectious processes mediated by the quorum sensing mechanism.
Assuntos
Hidrolases de Triester Fosfórico , Percepção de Quorum , Humanos , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Escherichia coli/metabolismo , Peróxido de Hidrogênio , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Biofilmes , Bactérias/metabolismo , Estabilidade EnzimáticaRESUMO
Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.
Assuntos
Hidrolases , Hidrolases de Triester Fosfórico , Acetilcolinesterase , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biocatálise , CatáliseRESUMO
A related group of phosphotriesters known as organophosphate flame retardants (OPFRs) has become emerging contaminants due to its worldwide use. The lack of an easily hydrolysable bond renders OPFRs inert to the well-known phosphotriesterases capable of hydrolyzing the neurotoxic organophosphates. An OPFRs phosphotriesterase gene stpte was cloned from plasmid pStJH of strain Sphingopyxis terrae subsp. terrae YC-JH3 and was heterologously expressed in Escherichia coli. The recombinant protein St-PTE was purified and analyzed. St-PTE showed the highest catalytic activity at pH 8.5 and 35 °C. The optimal substrate for St-PTE is triphenyl phosphate, with kcat/Km of 5.03 × 106 M-1 s-1, two orders of magnitude higher than those of tricresyl phosphate (4.17 × 104 M-1 s-1), 2-ethylhexyl diphenyl phosphate (2.03 × 104 M-1 s-1) and resorcinol bis(diphenyl phosphate) (6.30 × 104 M-1 s-1). St-PTE could break the P-O bond of tri-esters and convert aryl-OPFRs into their corresponding di-ester metabolites, including polymers of resorcinol bis(diphenyl phosphate). Mediated by transposase, the gene of OPFRs phosphotriesterase could be transferred horizontally among closely related strains of Sphingomonas, Sphingobium and Sphingopyxis. KEY POINTS: ⢠St-PTE from Sphingopyxis terrae subsp. terrae YC-JH3 could hydrolyze aryl-OPFRs. ⢠Metabolites of RBDPP hydrolyzed by phosphotriesterase were identified. ⢠St-PTE could hydrolyze the P-O cleavage of dimer and trimer of RBDPP. ⢠Phosphotriesterase genes transfer among Sphingomonadaceae mediated by transposase.
Assuntos
Retardadores de Chama , Hidrolases de Triester Fosfórico , Tritolil Fosfatos , Compostos de Bifenilo , Ésteres , Retardadores de Chama/metabolismo , Organofosfatos/metabolismo , Fosfatos , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Polímeros , Proteínas Recombinantes , Resorcinóis , Sphingomonadaceae , TransposasesRESUMO
Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.
Assuntos
Organismos Aquáticos , Bactérias , Poluentes Ambientais , Compostos Organofosforados , Hidrolases de Triester Fosfórico , Organismos Aquáticos/enzimologia , Bactérias/enzimologia , Biodegradação Ambiental , Poluentes Ambientais/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Oceano Índico , Mar Mediterrâneo , Compostos Organofosforados/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Fósforo/metabolismo , Água do Mar/microbiologiaRESUMO
Due to deadly toxicity and high environmental stability of the nerve agent VX, an efficient decontamination approach is desperately needed in tackling its severe threat to human security. The enzymatic destruction of nerve agents has been generally considered as one of the most effective ways, and here the hydrolysis of VX by phosphotriesterase (PTE) was investigated by extensive QM/MM and MM MD simulations. The hydrolytic cleavage of P-S by PTE is a two-step process with the free energy spans of 15.8 and 26.0 kcal mol-1 for the RP- and SP-enantiomer VX, respectively, and such remarkable stereospecificity of VX enantiomers in the enzymatic degradation is attributed to their conformational compatibility with the active pocket. The structurally less adaptive SP-enantiomer allows one additional water molecule to enter the binuclear zinc center and remarkably facilitates the release of the degraded product. Overall, the rate-limiting steps in the enzymatic degradation of VX by PTE involve the degraded product release of the RP-enantiomer and the enzymatic P-S cleavage of the SP-enantiomer. Further computational analysis on the mutation of selected residues also revealed that H257Y, H257D, H254Q-H257F, and L7ep-3a variants allow more water molecules to enter the active site, which improves the catalytic efficiency of PTE, as observed experimentally. The present work provides mechanistic insights into the stereoselective hydrolysis of VX by PTE and the activity manipulation through the active-site accessibility of water molecules, which can be used for the enzyme engineering to defeat chemical warfare agents.
Assuntos
Substâncias para a Guerra Química , Agentes Neurotóxicos , Hidrolases de Triester Fosfórico , Domínio Catalítico , Substâncias para a Guerra Química/química , Substâncias para a Guerra Química/metabolismo , Substâncias para a Guerra Química/toxicidade , Descontaminação , Humanos , Hidrólise , Compostos Organotiofosforados , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , ÁguaRESUMO
The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.
Assuntos
Substâncias para a Guerra Química/toxicidade , Compostos Organotiofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Animais , Caulobacteraceae/enzimologia , Inibidores da Colinesterase/toxicidade , Masculino , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Engenharia de Proteínas , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.
Assuntos
Caulobacteraceae/enzimologia , Agentes Neurotóxicos/metabolismo , Hidrolases de Triester Fosfórico/metabolismo , Catálise , Cromatografia Líquida , Hidrólise , Mutação , Agentes Neurotóxicos/química , Agentes Neurotóxicos/toxicidade , Hidrolases de Triester Fosfórico/genética , Estereoisomerismo , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.
Assuntos
Anticorpos Biespecíficos/imunologia , Organofosfatos/metabolismo , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Engenharia de Proteínas , Multimerização Proteica , Domínio Catalítico , Hidrolases de Triester Fosfórico/imunologia , Hidrolases de Triester Fosfórico/metabolismo , Estrutura Quaternária de Proteína , Eletricidade EstáticaRESUMO
The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1'-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb-PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, kcat/Km, of Sb-PTE toward the test phosphate triesters ranged from â¼10 to 105 M-1 s-1. The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.
Assuntos
Naftóis/química , Hidrolases de Triester Fosfórico/metabolismo , Sphingomonadaceae/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Hidrólise , Cinética , Naftóis/metabolismo , Fosfatos/química , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/genética , Estereoisomerismo , Especificidade por SubstratoRESUMO
Insertions and deletions (InDels) are frequently observed in natural protein evolution, yet their potential remains untapped in laboratory evolution. Here we introduce a transposon-based mutagenesis approach (TRIAD) to generate libraries of random variants with short in-frame InDels, and screen TRIAD libraries to evolve a promiscuous arylesterase activity in a phosphotriesterase. The evolution exhibits features that differ from previous point mutagenesis campaigns: while the average activity of TRIAD variants is more compromised, a larger proportion has successfully adapted for the activity. Different functional profiles emerge: (i) both strong and weak trade-off between activities are observed; (ii) trade-off is more severe (20- to 35-fold increased kcat/KM in arylesterase with 60-400-fold decreases in phosphotriesterase activity) and (iii) improvements are present in kcat rather than just in KM, suggesting adaptive solutions. These distinct features make TRIAD an alternative to widely used point mutagenesis, accessing functional innovations and traversing unexplored fitness landscape regions.
Assuntos
Mutação INDEL/genética , Evolução Molecular , Humanos , Mutagênese/genética , Mutagênese/fisiologia , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Biologia Sintética/métodosRESUMO
Organophosphorus (OP)1 nerve agents pose a severe toxicological threat, both after dissemination in military conflicts and by terrorists. Hydrolytic enzymes, which may be administered into the blood stream of victims by injection and can decompose the circulating nerve agent into non-toxic metabolites in vivo, could offer a treatment. Indeed, for the phosphotriesterase found in the bacterium Brevundimonas diminuta (BdPTE),2 engineered versions with improved catalytic efficiencies have been described; yet, their biochemical stabilities are insufficient for therapeutic use. Here, we describe the application of rational protein design to develop novel mutants of BdPTE that are less susceptible to oxidative damage. In particular, the replacement of two unpaired cysteine residues by more inert amino acids led to higher stability while maintaining high catalytic activity towards a broad spectrum of substrates, including OP pesticides and V-type nerve agents. The mutant BdPTE enzymes were produced in Escherichia coli, purified to homogeneity, and their biochemical and enzymological properties were assessed. Several candidates both revealed enhanced thermal stability and were less susceptible to oxidative stress, as demonstrated by mass spectrometry. These mutants of BdPTE may show promise for the treatment of acute intoxications by nerve agents as well as OP pesticides.
Assuntos
Antídotos/farmacologia , Proteínas de Bactérias/farmacologia , Caulobacteraceae/enzimologia , Agentes Neurotóxicos/intoxicação , Intoxicação por Organofosfatos/tratamento farmacológico , Compostos Organofosforados/toxicidade , Hidrolases de Triester Fosfórico/farmacologia , Antídotos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacteraceae/genética , Estabilidade de Medicamentos , Estabilidade Enzimática , Temperatura Alta , Mutação , Intoxicação por Organofosfatos/enzimologia , Compostos Organotiofosforados/intoxicação , Oxirredução , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Desnaturação Proteica , Proteínas Recombinantes/farmacologia , Sarina/intoxicação , Soman/intoxicaçãoRESUMO
Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents-GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme 'cocktail' comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.
Assuntos
Bactérias/enzimologia , Agentes Neurotóxicos/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/farmacologia , Antídotos/metabolismo , Antídotos/farmacologia , Bactérias/genética , Bactérias/metabolismo , Clonagem Molecular , Estabilidade Enzimática , Hidrolases de Triester Fosfórico/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Genes encoding structurally independent phosphotriesterases (PTEs) are identified in soil bacteria. These pte genes, often identified on mobilizable and self-transmissible plasmids are organized as mobile genetic elements. Their dissemination through lateral gene transfer is evident due to the detection of identical organophosphate degradation genes among soil bacteria with little orno taxonomic relationship. Convergent evolution of PTEs provided selective advantages to the bacterial strain as they convert toxic phosphotriesters (PTs) into a source of phosphate. The residues of organophosphate (OP) compounds that accumulate in a soil are proposed to contribute to the evolution of PTEs through substrate-assisted gain-of-function. This review provides comprehensive information on lateral transfer of pte genes and critically examines proposed hypotheses on their evolution in the light of the short half-life of OPs in the environment. The review also proposes alternate factors that have possibly contributed to the evolution and lateral mobility of PTEs by taking into account their biology and analyses of pte genes in genomic and metagenomic databases.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Organofosfatos/metabolismo , Hidrolases de Triester Fosfórico/genética , Solo/química , Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Evolução Molecular , Hidrolases de Triester Fosfórico/metabolismo , Plasmídeos , Homologia de SequênciaRESUMO
The bacterial enzyme phosphotriesterase (PTE) is noted for its ability to hydrolyze many organophosphate compounds, including insecticides and chemical warfare agents. PTE has been the subject of multiple enzyme evolution attempts, which have been highly successful against specific insecticides and the G-type nerve agents. Similar attempts targeting the V-type nerve agents have failed to achieve the same degree of success. Enzyme evolution is an inherently complex problem, which is complicated by synergistic effects, the need to use analogues in high-throughput screening, and a lack of quantitative data to direct future efforts. Previous evolution experiments with PTE have assumed an absence of synergy and minimally screened large libraries, which provides no quantitative information about the effects of individual mutations. Here a systemic approach has been applied to a 28800-member six-site PTE library. The library is screened against multiple V-agent analogues, and a combination of sequence and quantitative activity analysis is used to extract data about the effects of individual mutations. We demonstrate that synergistic relationships dominate the evolutionary landscape of PTE and that analogue activity profiles can be used to identify variants with high activity for substrates. Using these approaches, multiple variants with kcat/ Km values for the hydrolysis of VX that were improved >1500-fold were identified, including one variant that is improved 9200-fold relative to wild-type PTE and is specific for the SP enantiomer of VX. Multiple variants that were highly active for ( SP)-VR were identified, the best of which has a kcat/ Km values that is improved 13400-fold relative to that of wild-type PTE.
Assuntos
Proteínas de Bactérias/química , Substâncias para a Guerra Química/química , Compostos Organofosforados/química , Compostos Organotiofosforados/química , Hidrolases de Triester Fosfórico/química , Adaptação Fisiológica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Substâncias para a Guerra Química/metabolismo , Descontaminação , Evolução Molecular Direcionada , Hidrólise , Mutação , Organofosfatos/química , Organofosfatos/metabolismo , Compostos Organofosforados/metabolismo , Compostos Organotiofosforados/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Pseudomonas/enzimologia , Pseudomonas/genética , Estereoisomerismo , Especificidade por SubstratoRESUMO
Organophosphate compounds bioremediation by use of organophosphorus degradation enzymes such as DFPase is a developing interest in industry and medicine. The most important problem with the bio-catalytic enzymes is their instability on high temperatures. This work carried out to find suitable locations for introducing disulfide bridges in DFPase enzyme. We employed some computational approaches to design the disulfide bridges and evaluate their roles in the enzyme structural thermostability. According to the in silico results, mutant 6 (V24C, C76) increased the enzyme thermostability relative to wild-type.
Assuntos
Loligo/enzimologia , Hidrolases de Triester Fosfórico/química , Animais , Domínio Catalítico , Bases de Dados de Proteínas , Dissulfetos/química , Estabilidade Enzimática , Temperatura Alta , Loligo/química , Loligo/genética , Simulação de Dinâmica Molecular , Hidrolases de Triester Fosfórico/genética , Mutação Puntual , Conformação ProteicaRESUMO
Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.
Assuntos
Domínio Catalítico , Coenzima A Ligases/química , Hidrolases de Triester Fosfórico/química , Engenharia de Proteínas , Acil Coenzima A/biossíntese , Acil Coenzima A/química , Catálise , Coenzima A Ligases/genética , Cinética , Mutação , Compostos Organofosforados/química , Hidrolases de Triester Fosfórico/genética , Filogenia , Software , Especificidade por SubstratoRESUMO
Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.
Assuntos
Técnicas Biossensoriais/métodos , Substâncias para a Guerra Química/análise , Descontaminação/métodos , Agentes Neurotóxicos/análise , Compostos Organofosforados/análise , Compostos Organofosforados/metabolismo , Praguicidas/análise , Alicyclobacillus/enzimologia , Alicyclobacillus/genética , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Substâncias para a Guerra Química/metabolismo , Limite de Detecção , Agentes Neurotóxicos/metabolismo , Praguicidas/metabolismo , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Sulfolobus acidocaldarius/enzimologia , Sulfolobus acidocaldarius/genética , Sulfolobus solfataricus/enzimologia , Sulfolobus solfataricus/genéticaRESUMO
Automated design of enzymes with wild-type-like catalytic properties has been a long-standing but elusive goal. Here, we present a general, automated method for enzyme design through combinatorial backbone assembly. Starting from a set of homologous yet structurally diverse enzyme structures, the method assembles new backbone combinations and uses Rosetta to optimize the amino acid sequence, while conserving key catalytic residues. We apply this method to two unrelated enzyme families with TIM-barrel folds, glycoside hydrolase 10 (GH10) xylanases and phosphotriesterase-like lactonases (PLLs), designing 43 and 34 proteins, respectively. Twenty-one GH10 and seven PLL designs are active, including designs derived from templates with <25% sequence identity. Moreover, four designs are as active as natural enzymes in these families. Atomic accuracy in a high-activity GH10 design is further confirmed by crystallographic analysis. Thus, combinatorial-backbone assembly and design may be used to generate stable, active, and structurally diverse enzymes with altered selectivity or activity.
Assuntos
Técnicas de Química Combinatória , Glicosídeo Hidrolases/química , Hidrolases de Triester Fosfórico/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Cinética , Modelos Moleculares , Hidrolases de Triester Fosfórico/genética , Hidrolases de Triester Fosfórico/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
Metallo-ß-lactamase (MßL) is a eubacterial zinc metallo-hydrolase superfamily. Despite their well-known lactamase activities, MßL family members also have the ability to catalyze phosphotriester hydrolysis with different phosphotriesterase activities. In the present study, based on crystal structure comparisons of the related MßL members, a series of models was constructed and calculated using the density functional theory (DFT) method to explore the relationship between active-site changes and phosphotriesterase activities. These calculations show that the energetic barriers for phosphotriesterase activity are considerably reduced due to active-site differences, which describes an evolutionary trend for the development of phosphotriesterase activity in the MßL superfamily. The key event is the appearance of a specialized and negatively charged residue bridging both zinc ions, which plays the two important roles of maintaining charge balance and stabilizing the binuclear active-site structure. This pathway is also consistent with the evolutionary relationships determined by phylogenetic tree analysis using complete residue sequences. Our studies provide the first methodology to explore the development of a new enzyme activity within a superfamily, and to shed new light on understanding the catalytic mechanism from an evolutionary perspective.