RESUMEN
In the accompanying paper, we described evolving a lipase to the point where variants were soluble, stable and capable of degrading C8 TAG and C8 esters. These variants were tested for their ability to survive in an environment that might be encountered in a washing machine. Unfortunately, they were inactivated both by treatment with a protease used in laundry detergents and by very low concentrations of sodium dodecyl sulfate (SDS). In addition, all the variants had very low levels of activity with triglycerides with long aliphatic chains and with naturally occurring oils, like olive oil. Directed evolution was used to select variants with enhanced properties. In the first 10 rounds of evolution, the primary screen was selected for variants capable of hydrolyzing olive oil whereas the secondary screen was selected for enhanced tolerance towards a protease and SDS. In the final six rounds of evolution, the primary and secondary screens identified variants that retained activity after treatment with SDS. Sixteen cycles of evolution gave variants with greatly enhanced lipolytic activity on substrates that had both long (C16 and C18) as well as short (C3 and C8) chains. We found variants that were stable for more than 3 hours in protease concentrations that rapidly degrade the wild-type enzyme. Enhanced tolerance towards SDS was found in variants that could break down naturally occurring lipid and resist protease attack. The amino acid changes that gave enhanced properties were concentrated in the cap domain responsible for substrate binding.
Asunto(s)
Evolución Molecular Dirigida , Lipasa/genética , Lipasa/metabolismo , Péptido Hidrolasas/metabolismo , Ingeniería de Proteínas , Triglicéridos/metabolismo , Detergentes/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/genética , Hidrólisis , Lipasa/química , Proteolisis , Dodecil Sulfato de Sodio/farmacología , Solubilidad , Especificidad por Sustrato , TemperaturaRESUMEN
An enzyme must be soluble, stable, active and easy to produce to be useful in industrial applications. Not all enzymes possess these attributes. We set out to determine how many changes are required to convert an enzyme with poor properties into one that has useful properties. Lipase Lip3 from Drosophila melanogaster had been previously optimised for expression in Escherichia coli. The expression levels were good, but Lip3 was mainly insoluble with poor activity. Directed evolution was used to identify variants with enhanced activity along with improved solubility. Five variants and the wild-type (wt) enzyme were purified and characterised. The yield of the wt enzyme was just 2.2 mg/L of culture, while a variant, produced under the same conditions, gave 351 mg. The improvement of activity of the best variant was 200 times higher than that of the wt when the crude lysates were analysed using pNP-C8, but with purified protein, the improvement observed was 1.5 times higher. This means that most of the increase of activity is due to increase in solubility and stability. All the purified variants showed increased thermal stability compared with the wt enzyme that had a T1/2 of 37°C, while the mutant with P291L of 42.2°C and the mutant R7_47D with five mutations had a value of 52.9°C, corresponding to an improvement of 16°C. The improved variants had between five and nine changes compared with the wt enzyme. There were four changes that were found in all 30 final round variants for which sequences were obtained; three of these changes were found in the substrate-binding domain.
Asunto(s)
Evolución Molecular Dirigida , Proteínas de Drosophila/química , Lipasa/química , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Estabilidad de Enzimas/genética , Lipasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Metal ion-dependent, organophosphate-degrading enzymes (OP hydrolases) have received increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin and VX. These enzymes thus garner strong potential as bioremediators. The OP hydrolase from Agrobacterium radiobacter (OpdA) is one of the most efficient members of this group of enzymes. Previous studies have indicated that the choice of the hydrolysis-initiating nucleophile may depend on the pH of the reaction, with a metal ion-bridging hydroxide being preferred at lower pH (i.e. pH≤8.5), and a terminally coordinated hydroxide at higher pH (i.e. pH>9.0). Furthermore, fluoride was shown to be a potent inhibitor of the reaction, but only at low pH. Here, the crystal structure (1.3Å, pH6) of OpdA in presence of fluoride is described. While the first coordination sphere in the active site displays minimal changes in the presence of fluoride, the hydrogen bonding network that connects the dimetallic metal center to the substrate binding pocket is disrupted. Thus, the structure of fluoride-inhibited OpdA demonstrates the significance of this hydrogen bond network in controlling the mechanism and function of this enzyme.
Asunto(s)
Monoéster Fosfórico Hidrolasas/química , Agrobacterium tumefaciens , Dominio Catalítico , Cobalto/química , Complejos de Coordinación/química , Cristalografía por Rayos X , Fluoruros/farmacología , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Estructura Molecular , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Conformación ProteicaRESUMEN
Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cobalto/metabolismo , Enterobacter aerogenes/enzimología , Manganeso/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Sustitución de Aminoácidos , Asparagina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Calorimetría , Dominio Catalítico , Activación Enzimática , Enlace de Hidrógeno , Cinética , Mutación , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Fósforo/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina/química , Termodinámica , VolumetríaRESUMEN
Metallo-ß-lactamases (MBLs) with activity towards a broad-spectrum of ß-lactam antibiotics have become a major threat to public health, not least due to their ability to rapidly adapt their substrate preference. In this study, the capability of the MBL AIM-1 to evade antibiotic pressure by introducing specific mutations was probed by two alternative methods, i.e. site-saturation mutagenesis (SSM) of active site residues and in vitro evolution. Both approaches demonstrated that a single mutation in AIM-1 can greatly enhance a pathogen's resistance towards broad spectrum antibiotics without significantly compromising the catalytic efficiency of the enzyme. Importantly, the evolution experiments demonstrated that relevant amino acids are not necessarily in close proximity to the catalytic centre of the enzyme. This observation is a powerful demonstration that MBLs have a diverse array of possibilities to adapt to new selection pressures, avenues that cannot easily be predicted from a crystal structure alone.
Asunto(s)
Evolución Biológica , Farmacorresistencia Microbiana , Antibacterianos/química , Antibacterianos/farmacología , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular Dirigida , Ingeniería Genética , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida , Mutación/genética , Especificidad por Sustrato/efectos de los fármacos , beta-Lactamas/química , beta-Lactamas/farmacologíaRESUMEN
Antibiotic resistance has emerged as a major threat to global health care. This is largely due to the fact that many pathogens have developed strategies to acquire resistance to antibiotics. Metallo-ß-lactamases (MBL) have evolved to inactivate most of the commonly used ß-lactam antibiotics. AIM-1 is one of only a few MBLs from the B3 subgroup that is encoded on a mobile genetic element in a major human pathogen. Here, its mechanism of action was characterised with a combination of spectroscopic and kinetic techniques and compared to that of other MBLs. Unlike other MBLs it appears that AIM-1 has two avenues available for the turnover of the substrate nitrocefin, distinguished by the identity of the rate-limiting step. This observation may be relevant with respect to inhibitor design for this group of enzymes as it demonstrates that at least some MBLs are very flexible in terms of interactions with substrates and possibly inhibitors.
Asunto(s)
Antibacterianos/química , Aurora Quinasa B/química , Cefalosporinas/química , beta-Lactamasas/química , Aurora Quinasa B/metabolismo , Humanos , Cinética , Especificidad por SustratoRESUMEN
Enzymes have the potential to catalyse a wide variety of chemical reactions. They are increasingly being sought as environmentally friendly and cost-effective alternatives to conventional catalysts used in industries ranging from bioremediation to applications in medicine and pharmaceutics. Despite the benefits, they are not without their limitations. Many naturally occurring enzymes are not suitable for use outside of their native cellular environments. However, protein engineering can be used to generate enzymes tailored for specific industrial applications. Directed evolution is particularly useful and can be employed even when lack of structural information impedes the use of rational design. The aim of this review is to provide an overview of current industrial applications of enzyme technology and to show how directed evolution can be used to modify and to enhance enzyme properties. This includes a brief discussion on library generation and a more detailed focus on library screening methods, which are critical to any directed evolution experiment.
Asunto(s)
Evolución Molecular Dirigida , Enzimas/metabolismo , Biocatálisis , Productos Biológicos/metabolismo , Enzimas/química , Enzimas/genética , Mutagénesis , Preparaciones Farmacéuticas/metabolismo , Ingeniería de ProteínasRESUMEN
The diesterase Rv0805 from Mycobacterium tuberculosis is a dinuclear metallohydrolase that plays an important role in signal transduction by controlling the intracellular levels of cyclic nucleotides. As Rv0805 is essential for mycobacterial growth it is a promising new target for the development of chemotherapeutics to treat tuberculosis. The in vivo metal-ion composition of Rv0805 is subject to debate. Here, we demonstrate that the active site accommodates two divalent transition metal ions with binding affinities ranging from approximately 50â nm for Mn(II) to about 600â nm for Zn(II) . In contrast, the enzyme GpdQ from Enterobacter aerogenes, despite having a coordination sphere identical to that of Rv0805, binds only one metal ion in the absence of substrate, thus demonstrating the significance of the outer sphere to modulate metal-ion binding and enzymatic reactivity. Ca(II) also binds tightly to Rv0805 (Kd ≈40â nm), but kinetic, calorimetric, and spectroscopic data indicate that two Ca(II) ions bind at a site different from the dinuclear transition-metal-ion binding site. Ca(II) acts as an activator of the enzymatic activity but is able to promote the hydrolysis of substrates even in the absence of transition-metal ions, thus providing an effective strategy for the regulation of the enzymatic activity.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Calcio/química , Iones/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Elementos de Transición/química , Sitios de Unión , Unión ProteicaRESUMEN
Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.
Asunto(s)
Organofosfatos/química , Plaguicidas/química , Monoéster Fosfórico Hidrolasas/química , Cloropirifos/análogos & derivados , Cloropirifos/química , Barajamiento de ADN , Evolución Molecular Dirigida , Hidrólisis , Cinética , Metil Paratión/química , Modelos Moleculares , Mutación , Paraoxon/análogos & derivados , Paraoxon/química , Paratión/química , Monoéster Fosfórico Hidrolasas/genética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3ß,17ß-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation.
Asunto(s)
Arilsulfatasas/metabolismo , Pseudomonas aeruginosa/enzimología , Esteroides/metabolismo , Sulfatos/metabolismo , Pruebas de Enzimas/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Hidrólisis , Esteroides/análisis , Esteroides/orina , Sulfatos/análisis , Sulfatos/orinaRESUMEN
Directed evolution is a common tool employed to generate enzymes suitable for industrial use. High thermal stability is often advantageous or even a requirement for biocatalysts, as such the evolution of protein stability is of practical as well as academic interest. Even when evolving enzymes for new or improved catalytic functions, stability is an important factor since it can limit the accumulation rate and number of desired active site mutations. Dienelactone hydrolase, a small monomeric protein, has been previously evolved via a three-stage process to possess enhanced activity and specificity toward non-physiological substrates. In addition to seven active site mutations there were three surface mutations that were thought to increase the stability of the enzyme and compensate for the destabilizing active site mutations. Here, the individual influence of the three surface mutations--Q110L, Y137C and N154D--on the thermal and chemical stability of DLH has been assessed. While the Q110L and N154D mutations improved the thermal stability, the influence of the Y137C mutation was more complex. Individually it was destabilizing both thermally and chemically, but when in the presence of the Q110L and N154D mutations its effect was neutralized in relation to thermal but not chemical stability. In the context of a directed evolution experiment, these compensatory surface mutations play important roles. However, our results show that detrimental mutations can arise, thus the simultaneous monitoring of stability changes while evolving enzymes for enhanced catalytic properties can be beneficial.
Asunto(s)
Sustitución de Aminoácidos , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Evolución Molecular Dirigida , Mutación Missense , Estabilidad de Enzimas/genética , Estructura Secundaria de ProteínaRESUMEN
The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/ß hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Evolución Molecular Dirigida/métodos , Bioingeniería , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Cristalización , Cinética , Modelos Moleculares , Nitrofenoles/metabolismo , Conformación Proteica , Pliegue de Proteína , Bibliotecas de Moléculas Pequeñas , Especificidad por SustratoRESUMEN
Metal ion-dependent, organophosphate-degrading enzymes have acquired increasing attention due to their ability to degrade and thus detoxify commonly used pesticides and nerve agents such as sarin. The best characterized of these enzymes are from Pseudomonas diminuta (OPH) and Agrobacterium radiobacter (OpdA). Despite high sequence homology (>90 % identity) and conserved metal ion coordination these enzymes display considerable variations in substrate specificity, metal ion affinity/preference and reaction mechanism. In this study, we highlight the significance of the presence (OpdA) or absence (OPH) of an extended hydrogen bond network in the active site of these enzymes for the modulation of their catalytic properties. In particular, the second coordination sphere residue in position 254 (Arg in OpdA, His in OPH) is identified as a crucial factor in modulating the substrate preference and binding of these enzymes. Inhibition studies with fluoride also support a mechanism for OpdA whereby the identity of the hydrolysis-initiating nucleophile changes as the pH is altered. The same is not observed for OPH.
Asunto(s)
Agrobacterium tumefaciens/enzimología , Inhibidores Enzimáticos/farmacología , Fluoruros/farmacología , Organofosfatos/farmacología , Hidrolasas de Triéster Fosfórico/antagonistas & inhibidores , Pseudomonas/enzimología , Agrobacterium tumefaciens/aislamiento & purificación , Calorimetría , Inhibidores Enzimáticos/química , Fluoruros/química , Concentración de Iones de Hidrógeno , Cinética , Organofosfatos/química , Hidrolasas de Triéster Fosfórico/metabolismo , Pseudomonas/aislamiento & purificación , Relación Estructura-Actividad , Especificidad por Sustrato , TermodinámicaRESUMEN
Interleukin-3 (IL-3) is a cytokine secreted by mast cells and activated T-cells known to be an important regulator of differentiation, survival, proliferation and activation of a range of haemopoietic lineages. The effects of IL-3 on target cells are mediated by a transmembrane receptor system composed of a cytokine-specific α-subunit and a ß-subunit, the principal signalling entity. In the mouse, two ß-subunits have co-evolved: a common ß-subunit (ßc) shared between IL-3 and the related cytokines IL-5 and granulocyte/macrophage colony-stimulating factor (GM-CSF); and an IL-3-specific ß-subunit (ßIL-3). ßIL-3 differs from ßc in its specificity for IL-3 and its capacity to bind IL-3 directly in the absence of an α-subunit, and, in the absence of structural information, the basis for these properties has remained enigmatic. In the present study, we have solved the crystal structure of the ßIL-3 ectodomain at 3.45 Å (1 Å=0.1 nm) resolution. This structure provides the first evidence that ßIL-3 adopts an arch-shaped intertwined homodimer with similar topology to the paralogous ßc structure. In contrast with apo-ßc, however, the ligand-binding interface of ßIL-3 appears to pre-exist in a conformation receptive to IL-3 engagement. Molecular modelling of the IL-3-ßIL-3 interface, in conjunction with previous mutational studies, suggests that divergent evolution of both ßIL-3 and IL-3 underlies their unique capacity for direct interaction and specificity.
Asunto(s)
Subunidad beta Común de los Receptores de Citocinas/química , Interleucina-3/química , Animales , Cristalografía por Rayos X , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Evolución Molecular , Interleucina-3/metabolismo , Ratones , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
Dienelactone hydrolase (DLH) is a monomeric protein with a simple α/ß-hydrolase fold structure. It readily crystallizes in space group P212121 from either a phosphate or ammonium sulfate precipitation buffer. Here, the structure of DLH at 1.85â Å resolution crystallized in space group C2 with two molecules in the asymmetric unit is reported. When crystallized in space group P212121 DLH has either phosphates or sulfates bound to the protein in crucial locations, one of which is located in the active site, preventing substrate/inhibitor binding. Another is located on the surface of the enzyme coordinated by side chains from two different molecules. Crystallization in space group C2 from a sodium citrate buffer results in new crystallographic protein-protein interfaces. The protein backbone is highly similar, but new crystal contacts cause changes in side-chain orientations and in loop positioning. In regions not involved in crystal contacts, there is little change in backbone or side-chain configuration. The flexibility of surface loops and the adaptability of side chains are important factors enabling DLH to adapt and form different crystal lattices.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas de Éster Carboxílico/química , Pseudomonas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Citratos/química , Cristalografía por Rayos X , Modelos Moleculares , Fosfatos/química , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Pseudomonas/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Citrato de Sodio , Sulfatos/químicaRESUMEN
A library of steroid glucuronides was prepared using the glucuronylsynthase derived from Escherichia coliß-glucuronidase, followed by purification using solid-phase extraction. A representative range of steroid substrates were screened for synthesis on the milligram scale under optimised conditions with conversions dependent on steroid substitution and stereochemistry. Epiandrosterone (3ß-hydroxy-5α-androstan-17-one) provided the highest conversion of 90% (84% isolated yield). The previously unreported glucuronide conjugates of methandriol (17α-methylandrost-5-ene-3ß,17ß-diol), cholest-5-ene-3ß,25-diol and the designer steroid trenazone (17ß-hydroxyestra-4,9-dien-3-one) were prepared on a multi-milligram scale suitable for characterisation by (1)H and (13)C NMR spectroscopy. The glucuronide conjugate of d5-etiocholanolone (2,2,3,4,4-d5-3α-hydroxy-5ß-androstan-17-one), a target developed by the World Anti-Doping Agency as a certified reference material, was also prepared on a milligram scale. The improved E. coli glucuronylsynthase method provides for the rapid synthesis and purification of steroid glucuronides on a scale suitable for a range of analytical applications.
Asunto(s)
Escherichia coli/enzimología , Glucuronidasa/metabolismo , Glucurónidos/biosíntesis , Esteroides/biosíntesis , Espectroscopía de Resonancia Magnética con Carbono-13 , Glucurónidos/química , Espectroscopía de Protones por Resonancia Magnética , Esteroides/químicaRESUMEN
Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate-the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.
Asunto(s)
Calorimetría/métodos , Hidrolasas/metabolismo , Metaloproteasas/metabolismo , Catálisis , Conductometría/métodos , Hidrolasas/química , Metaloproteasas/química , Paraoxon/análisis , Paraoxon/química , Paraoxon/metabolismoRESUMEN
Organophosphorus (OP) pesticides are a diverse class of acetylcholinesterase (AChE) inhibitors that are responsible for tremendous morbidity and mortality worldwide, killing approximately 300,000 people annually. Enzymatic hydrolysis of OPs is a potential therapy for acute poisoning. OpdA, an OP hydrolase isolated from Agrobacterium radiobacter, has been shown to decrease lethality in rodent models of OP poisoning. This study investigated the effects of OpdA on AChE activity, plasma concentrations of OP, and signs of toxicity after administration of dichlorvos to nonhuman primates. A dose of 75 mg/kg dichlorvos given orally caused apnea within 10 min with a progressive decrease in heart rate. Blood AChE activity decreased to zero within 10 min. Respirations and AChE activity did not recover. The mean dichlorvos concentration rose to a peak of 0.66 µg/ml. Treated monkeys received 1.2mg/kg OpdA iv immediately after poisoning with dichlorvos. In Opda-treated animals, heart and respiratory rates were unchanged from baseline over a 240-minute observation period. AChE activity slowly declined, but remained above 25% of baseline for the entire duration. Dichlorvos concentrations reached a mean peak of 0.19 µg/ml at 40 min after poisoning and decreased to a mean of 0.05 µg/ml at 240 min. These results show that OpdA hydrolyzes dichlorvos in an African green monkey model of lethal poisoning, delays AChE inhibition, and prevents lethality.
Asunto(s)
Agrobacterium/enzimología , Antídotos/uso terapéutico , Proteínas Bacterianas/uso terapéutico , Modelos Animales de Enfermedad , Hidrolasas/uso terapéutico , Intoxicación por Organofosfatos/tratamiento farmacológico , Plaguicidas/metabolismo , Acetilcolinesterasa/sangre , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Antídotos/aislamiento & purificación , Antídotos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Chlorocebus aethiops , Inhibidores de la Colinesterasa/sangre , Inhibidores de la Colinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacocinética , Inhibidores de la Colinesterasa/toxicidad , Depresión Química , Diclorvos/sangre , Diclorvos/metabolismo , Diclorvos/farmacocinética , Diclorvos/toxicidad , Eritrocitos/efectos de los fármacos , Eritrocitos/enzimología , Frecuencia Cardíaca/efectos de los fármacos , Hidrolasas/genética , Hidrolasas/aislamiento & purificación , Hidrolasas/metabolismo , Hidrólisis/efectos de los fármacos , Masculino , Intoxicación por Organofosfatos/fisiopatología , Plaguicidas/sangre , Plaguicidas/farmacocinética , Plaguicidas/toxicidad , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Frecuencia Respiratoria/efectos de los fármacos , Índice de Severidad de la Enfermedad , Especificidad por Sustrato , Análisis de SupervivenciaRESUMEN
An enhanced understanding of the metal ion binding and active site structural features of phosphoesterases such as the glycerophosphodiesterase from Enterobacter aerogenes (GpdQ), and the organophosphate degrading agent from Agrobacterium radiobacter (OpdA) have important consequences for potential applications. Coupled with investigations of the metalloenzymes, programs of study to synthesise and characterise model complexes based on these metalloenzymes can add to our understanding of structure and function of the enzymes themselves. This review summarises some of our work and illustrates the significance and contributions of model studies to knowledge in the area.
Asunto(s)
Materiales Biocompatibles/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Agrobacterium tumefaciens/enzimología , Materiales Biocompatibles/síntesis química , Dominio Catalítico , Complejos de Coordinación/síntesis química , Complejos de Coordinación/metabolismo , Enterobacter aerogenes/enzimología , Metales/química , Metales/metabolismo , Organofosfatos/química , Organofosfatos/metabolismo , Hidrolasas Diéster Fosfóricas/química , Unión ProteicaRESUMEN
Metallo-ß-lactamases (MBLs) are a family of metalloenzymes that are capable of hydrolyzing ß-lactam antibiotics and are an important means by which bacterial pathogens use to inactivate antibiotics. A database search of the available amino acid sequences from Serratia proteamaculans indicates the presence of an unusual MBL. A full length amino acid sequence alignment indicates overall homology to B3-type MBLs, but also suggests considerable variations in the active site, notably among residues that are relevant to metal ion binding. Steady-state kinetic measurements further indicate functional differences and identify two relevant pK a values for catalysis (3.8 for the enzyme-substrate complex and 7.8 for the free enzyme) and a preference for penams with modest reactivity towards some cephalosporins. An analysis of the metal ion content indicates the presence of only one zinc ion per active site in the resting enzyme. In contrast, kinetic data suggest that the enzyme may operate as a binuclear enzyme, and it is thus proposed that a catalytically active di-Zn(2+) center is formed only once the substrate is present.