RESUMEN
The highly cytotoxic macrocyclic trichothecene Isororidin A (C29H40O9) was isolated from the fungus Myrothesium verrucaria endophytic on the wild medicinal plant `Datura' (Datura stramonium L.) and was characterized by one- (1D) and two-dimensional (2D) NMR spectroscopy. The three-dimensional structure of Isororidin A has been confirmed by X-ray crystallography at 0.81â Å resolution from crystals grown in the orthorhombic space group P212121, with one molecule per asymmetric unit. Isororidin A is the epimer of previously described (by X-ray crystallography) Roridin A at position C-13' of the macrocyclic ring.
Asunto(s)
Tricotecenos , Cristalografía por Rayos X , Tricotecenos/química , Estructura MolecularRESUMEN
Research on glucuronoyl esterases (GEs) has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA) is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.
Asunto(s)
Esterasas/química , Ácido Glucurónico/química , Ésteres , Cinética , Especificidad por SustratoRESUMEN
The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-ß-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/ß-hydrolase fold with a three-layer αßα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.
Asunto(s)
Esterasas/química , Proteínas Fúngicas/química , Sporothrix/enzimología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Biocatálisis , Esterasas/genética , Proteínas Fúngicas/genética , Glucuronatos/química , Hidrólisis , Datos de Secuencia Molecular , Proteínas de Unión al ARN/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de Schizosaccharomyces pombe/química , Sporothrix/genética , Especificidad por SustratoRESUMEN
5-(O-Perbenzoylated-ß-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-ß-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-ß-D-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid-DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN(3). These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, ß-D-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, ß-D-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and ß-D-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(ß-D-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K(i)=854µM, 2-(ß-D-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K(i)=745µM).
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores Enzimáticos/síntesis química , Glicoconjugados/síntesis química , Glucógeno Fosforilasa de Forma Muscular/antagonistas & inhibidores , Fosforilasa b/antagonistas & inhibidores , Alquinos/química , Animales , Azidas/química , Catálisis , Diabetes Mellitus Tipo 2/fisiopatología , Inhibidores Enzimáticos/farmacología , Glucosa/química , Glicoconjugados/farmacología , Glucógeno Fosforilasa de Forma Muscular/metabolismo , Humanos , Cinética , Oxadiazoles/química , Fosforilasa b/metabolismo , Propionatos/química , Conejos , Triazoles/químicaRESUMEN
Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(ß-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3µM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the ß-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the ß-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.
Asunto(s)
Inhibidores Enzimáticos/química , Glucosa/química , Glucógeno Fosforilasa de Forma Muscular/antagonistas & inhibidores , Tiosemicarbazonas/química , Animales , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucofosfatos/química , Glucógeno Fosforilasa de Forma Muscular/metabolismo , Halógenos/química , Cinética , Conformación Molecular , Unión Proteica , Piridinas/química , Conejos , Tiosemicarbazonas/síntesis química , Tiosemicarbazonas/farmacologíaRESUMEN
O-peracetylated 1-(beta-D-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated beta-D-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-beta-D-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(beta-D-glucopyranosyl)-3,5-diphenyl- as well as 3-(beta-D-glucopyranosyl)-1,5-diphenyl biurets besides 1-(beta-D-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(beta-D-glucopyranosyl)-5-(beta-D-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated beta-D-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of beta-D-gluco, beta-D-galacto and beta-D-xylo configurations. O-Acyl protected 1-(beta-D-glucopyranosyl)-3-(beta-D-glycopyranosylcarbonyl)ureas were obtained from the reaction of beta-D-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of beta-D-gluco and beta-D-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary alpha-amylase.