RESUMEN
Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (ß = 0.0090 ± 0.0009 mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.
Asunto(s)
Acetofenonas/farmacología , Endo-1,4-beta Xilanasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Polisacáridos/metabolismo , Hidrólisis/efectos de los fármacos , CinéticaRESUMEN
Antibiotic-resistant bacteria (ARB) have been surveyed widely in water bodies, but few studies have determined the diversity of ARB in sediment, which is the most taxon-abundant habitat in aquatic environments. We isolated 56 extended-spectrum beta-lactamase (ESBL)-producing bacteria from a single sediment sample taken from an urban river in China. All strains were confirmed for ESBL-producing capability by both the clavulanic acid combination disc method and MIC determination. Of the isolated strains, 39 were classified as Enterobacteriaceae (consisting of the genera Escherichia, Klebsiella, Serratia, and Aeromonas) by 16S rRNA gene sequencing and biochemical analysis. The present study identifies, for the first time, ESBL-producing strains from the families Brucellaceae and Moraxellaceae. The bla(CTX-M) gene was the most dominant of the ESBL genes (45 strains), while the bla(TEM) gene was the second-most dominant (22 strains). A total of five types of bla(CTX-M) fragments were identified, with both known and novel sequences. A library of bla(CTX-M) cloned from the sediment DNA showed an even higher diversity of bla(CTX-M) sequences. The discovery of highly diverse ESBL-producing bacteria and ESBL genes, particularly bla(CTX), in urban river sediment raises alarms for potential dissemination of ARB in communities through river environments.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/genética , Variación Genética , Sedimentos Geológicos/microbiología , Ríos/microbiología , beta-Lactamasas/genética , Antibacterianos/farmacología , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , China , Ácido Clavulánico/farmacología , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Ecosistema , Pruebas de Sensibilidad Microbiana/métodos , Datos de Secuencia Molecular , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , beta-Lactamas/farmacologíaRESUMEN
In this study we conducted serial kinetic studies integrated with computational simulations to judge the inhibitory effect of pyrogallol on α-glucosidase, due to the association between this enzyme and the treatment of type 2 diabetes. As a result, we found that pyrogallol bound to the active site of α-glucosidase, interacting with several key residues, such as ASP68, MET69, TYR71, PHE157, PHE158, PHE177, GLN181, HIS348, ASP349, ASP406, VAL407, ASP408, ARG439, and ARG443, which was predicted by performing a protein-ligand docking simulation. Subsequently, we evaluated the inhibitory effect of pyrogallol on α-glucosidase, and found that it induced a mixed type of inhibition in a reversible and quick-binding manner. The relevant kinetic parameters were evaluated to be: IC50=0.72±0.051mM; Ki=0.37±0.018mM. A tertiary conformational change was synchronized with pyrogallol inhibition and modulation of the shape of the active site was correspondingly observed. Our study provides insight into the functional inhibitory role of pyrogallol, which results from its triple-hydroxyl groups interacting with the active site of α-glucosidase. We suggest that compounds similar to pyrogallol (phenolic hydroxyl compounds) which target the key residues of the active site of α-glucosidase could be potential agents for α-glucosidase inhibition.
Asunto(s)
Simulación del Acoplamiento Molecular , Pirogalol/farmacología , Saccharomyces cerevisiae/enzimología , alfa-Glucosidasas/farmacología , Sitios de Unión , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Cinética , Ligandos , Naftalenosulfonatos/química , Pirogalol/química , Espectrometría de Fluorescencia , Factores de Tiempo , alfa-Glucosidasas/químicaRESUMEN
Cadmium ions (Cd2+) are a widespread and easily absorbed toxin to both humans and animals that can be spread via food, water, and air pollution. Tyrosinase (EC 1.14.18.1) is a multifunctional copper-containing enzyme that is ubiquitously expressed in animals and plays a critical role in melanin production. We evaluated the toxic effects of Cd2+ on tyrosinase activity and conformation by measuring kinetics and computationally simulating the interactions. We found Cd2+ to be a slope-hyperbolic noncompetitive-inhibition reversible inhibitor of tyrosinase, with an IC50 of 2.92±0.16mM and Ki of 0.23±0.02mM. Spectrofluorimetric measurements of intrinsic and ANS-binding fluorescence showed that Cd2+ did not induce significant changes to tyrosinase overall or to its regional active site conformations. Cd2+ showed its inactivation effect not by modulating apparent structural changes to tyrosinase, but by occupying binding sites. To gain further insight into the Cd2+/tyrosinase interaction, we performed computational docking and molecular dynamics simulations. The results consistently indicated that Cd2+ can interact with several residues near the tyrosinase active site, primarily HIS85 and ASN260. Our study provides insight into the mechanism of the toxic effects Cd2+ has on tyrosinase, which could affect the normal pigmentation pathway in animals.
Asunto(s)
Cadmio/química , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Monofenol Monooxigenasa/química , Asparagina/química , Dominio Catalítico , Cationes Bivalentes , Histidina/química , Humanos , Cinética , Simulación de Dinámica Molecular , Monofenol Monooxigenasa/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/químicaRESUMEN
Due to the finding that inhibition of α-glucosidase is directly associated with treatment of several diseases, the development of a selective inhibitor for targeting α-glucosidase is important. Gallotannin (GT) is a natural ingredient that has been used as a food additive and for medicinal applications. In this study, we performed a computational docking experiment involving the pre-simulation of the binding mechanism of GT, and the effect of GT on α-glucosidase was evaluated with inhibitory kinetics based on its polyphenol properties. The computational simulations indicated that the hydroxyl groups of GT interact with several residues near the α-glucosidase active site (Met69, Tyr71, Phe177, Arg212, Asp214, Glu276, His348, Asp349, and Arg439), which could affect the catalytic function of α-glucosidase by retarding substrate access. Subsequent kinetic experiments showed that GT conspicuously inhibited α-glucosidase in a parabolic mixed-type manner (IC50=1.31±0.03µM;Ki=0.41±0.032µM). Our study provides insight into the inhibition mechanism and binding manner of GT to α-glucosidase. Based on its α-glucosidase-inhibiting effect and its demonstrated safety as a naturally derived compound, GT represents a promising potential agent for treatment of α-glucosidase-associated diseases.
Asunto(s)
Inhibidores de Glicósido Hidrolasas/farmacología , Taninos Hidrolizables/farmacología , alfa-Glucosidasas/metabolismo , Inhibidores de Glicósido Hidrolasas/metabolismo , Taninos Hidrolizables/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , alfa-Glucosidasas/químicaRESUMEN
α-Glucosidase is a critical metabolic enzyme that produces glucose molecules by catalyzing carbohydrates. The aim of this study is to elucidate biological toxicity of Cd(2+) based on α-glucosidase activity and conformational changes. We studied Cd(2+)-mediated inactivation as well as conformational modulation of α-glucosidase by using kinetics coupled with simulation of molecular dynamics. The enzyme was significantly inactivated by Cd(2+) in a reversibly binding behavior, and Cd(2+) binding induced a non-competitive type of inhibition reaction (the K i was calculated as 0.3863 ± 0.033 mM). Cd(2+) also modulated regional denaturation of the active site pocket as well as overall partial tertiary structural change. In computational simulations using molecular dynamics, simulated introduction of Cd(2+) induced in a depletion of secondary structure by docking Cd(2+) near the saccharides degradation at the active site, suggesting that Cd(2+) modulating enzyme denaturation. The present study elucidated that the binding of Cd(2+) triggers conformational changes of α-glucosidase as well as inactivates catalytic function, and thus suggests an explanation of the deleterious effects of Cd(2+) on α-glucosidase.
Asunto(s)
Cadmio/toxicidad , Contaminantes Ambientales/toxicidad , Inhibidores de Glicósido Hidrolasas/toxicidad , Saccharomyces cerevisiae/enzimología , alfa-Glucosidasas/metabolismo , Cinética , Simulación de Dinámica Molecular , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , alfa-Glucosidasas/químicaRESUMEN
Tyrosinase plays a central role in biological pigment formation, and hence knowledge of tyrosinase catalytic mechanisms and regulation may have medical, cosmetic, and agricultural applications. We found in this study that arabinose significantly inhibited tyrosinase, and this was accompanied by conformational changes in enzyme structure. Kinetic analysis showed that arabinose-mediated inactivation followed first-order kinetics, and single and multiple classes of rate constants were measured. Arabinose displayed a mixed-type inhibitory mechanism with K(i)=0.22±0.07 mM. Measurements of intrinsic and ANS-binding fluorescence showed that arabinose induced tyrosinase to unfold and expose inner hydrophobic regions. We simulated the docking between tyrosinase and arabinose (binding energies were -26.28 kcal/mol for Dock6.3 and -2.02 kcal/mol for AutoDock4.2) and results suggested that arabinose interacts mostly with His61, Asn260, and Met280. The present strategy of predicting tyrosinase inhibition by simulation of docking by hydroxyl groups may prove useful in screening for potential tyrosinase inhibitors, as shown here for arabinose.