RESUMEN
Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.
Asunto(s)
Lacasa , Polyporales , Hongos/metabolismo , Lacasa/genética , Lacasa/metabolismo , Lignina/metabolismo , Oxidación-Reducción , Polyporales/metabolismoRESUMEN
Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.
Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Antituberculosos/química , Aptámeros de Nucleótidos/química , Proteínas Bacterianas/antagonistas & inhibidores , ADN de Cadena Simple/química , Inhibidores Enzimáticos/química , Mycobacterium tuberculosis/efectos de los fármacos , Acetolactato Sintasa/química , Acetolactato Sintasa/genética , Animales , Antituberculosos/metabolismo , Antituberculosos/farmacología , Aptámeros de Nucleótidos/biosíntesis , Aptámeros de Nucleótidos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , ADN de Cadena Simple/biosíntesis , ADN de Cadena Simple/farmacología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Biblioteca de Genes , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Técnica SELEX de Producción de AptámerosRESUMEN
Acetohydroxyacid synthase (AHAS) catalyzes the first essential biosynthetic step of branched-chain amino acids and is a biologically safe target against Mycobacterium tuberculosis (MTB). In our previous research, we used virtual screening to identify some novel AHAS inhibitors as potent antituberculosis agents. In this study, we synthesized twenty-four additional quinazolinone benzoates and explored their antitubercular activity. Five of these compounds displayed significant MTB-AHAS inhibition and their IC50 values were determined to be in the range of 6.50 µM-12.08 µM. Importantly, these compounds also exhibited strong in vitro activity (MICs in the range of 2.5-10 mg/L) and intracellular activity against clinically isolated extensively drug-resistant strains of M. tuberculosis. Taken together, these results indicated that the quinazolinone benzoate compounds should be regarded as promising lead compounds for the development of potent antituberculosis drugs with a novel mode of action.
Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Antituberculosos/farmacología , Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Quinazolinonas/farmacología , Acetolactato Sintasa/metabolismo , Antituberculosos/síntesis química , Antituberculosos/química , Benzoatos/síntesis química , Benzoatos/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS.
Asunto(s)
Acetolactato Sintasa/genética , Aminoácidos/genética , Enterococcus faecalis/enzimología , Flavina-Adenina Dinucleótido/metabolismo , Mutagénesis Sitio-Dirigida/métodos , Acetolactato Sintasa/química , Acetolactato Sintasa/aislamiento & purificación , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Electroforesis en Gel de Poliacrilamida , Cinética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Ácido Pirúvico/metabolismo , Alineación de Secuencia , Espectrometría de Fluorescencia , Homología Estructural de Proteína , Tiamina Pirofosfato/metabolismo , Triptófano/metabolismoRESUMEN
Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.
Asunto(s)
Aptámeros de Nucleótidos/química , ADN de Cadena Simple/química , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación , Secuencia de Bases , Cartilla de ADN , Límite de Detección , Reacción en Cadena de la Polimerasa , Técnica SELEX de Producción de AptámerosRESUMEN
Mycobacterium tuberculosis AHAS is a potential target for the development of novel anti-tuberculosis agents. Silico analysis showed that conserved His84 and Gln86 residues lie in the catalytic dimer interface of M. tuberculosis AHAS. Mutational analyses of these invariants led to significant reduction in their activity with reduced affinity toward the substrate. Importantly, mutation of Gln86 to Trp abolished complete activity. Further, molecular dynamics simulation studies suggested that these residues are likely to play a key role in maintaining the Glu85 side chain in the required geometry with N1' atom of ThDP during catalysis. In addition, substitution of essential Glu85 by Ala, Asp, and Gln led to severe drop in catalytic activity with reduced affinity toward ThDP confirming its catalytic role in M. tuberculosis AHAS.
Asunto(s)
Acetolactato Sintasa/química , Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Catálisis , Secuencia Conservada , Dimerización , Escherichia coli/metabolismo , Ácido Glutámico/química , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Tiamina Pirofosfato/metabolismoRESUMEN
Acetohydroxyacid synthase (AHAS) of Mycobacterium tuberculosis is a promising target for the development of anti-tuberculosis agents. With the absence of an available bacterial AHAS crystal structure, that of M. tuberculosis, site-directed mutagenesis has been a useful tool for determining its structural and functional features. In this study, a highly conserved proline residue (P126 of M. tuberculosis AHAS) was selected, and the possible role was evaluated by site-directed mutagenesis. P126 was replaced by valine, threonine, alanine, and glutamate to yield P126V, P126T, P126A, and P126E, respectively. All variants were expressed in their soluble forms in Escherichia coli and purified to near homogeneity. The molecular mass (SDS-PAGE) of the purified variants was â¼68 kDa, which is similar to that of wild-type AHAS. The P126V, P126T, and P126A variants exhibited significantly lower activity than wild-type AHAS, whereas P126E was inactive under the tested assay conditions. Furthermore, the P126V and P126T variants showed a significantly decreased preference toward pyruvate and ThDP as substrate and cofactor respectively, whereas the P126A showed similar kinetics to that of wild-type AHAS. Like in AHAS from yeast Saccharomyces cerevisiae (PDB ID: 1N0H), residue P126 is located in the ThDP binding pocket of M. tuberculosis AHAS homology model. Collectively, these results suggest that the conserved P126 plays a significant role in the ThDP binding of M. tuberculosis AHAS.
Asunto(s)
Acetolactato Sintasa/metabolismo , Mycobacterium tuberculosis/enzimología , Tiamina Pirofosfato/metabolismo , Acetolactato Sintasa/química , Acetolactato Sintasa/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico/genética , Secuencia Conservada , Genes Bacterianos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Prolina/química , Conformación Proteica , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 µM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 µM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 µM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 µg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.
Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Pseudomonas aeruginosa/enzimología , Acetolactato Sintasa/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , TemperaturaRESUMEN
Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3µM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1µM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.