RESUMEN
The recent classification of glycoside hydrolase family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, carboxymethylcellulase, and Cel5A), from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that carboxymethylcellulose and phosphoric acid-swollen cellulose are in fact relatively poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage ß-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo-form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs that engender predominant mixed-linkage endo-glucanase activity vis à vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of (meta)genomes to elucidate the biology of complex gut ecosystems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Endo-1,3(4)-beta-Glucanasa/metabolismo , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Prevotella/enzimología , Sustitución de Aminoácidos , Apoenzimas/antagonistas & inhibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Celulasa/antagonistas & inhibidores , Celulasa/química , Celulasa/genética , Celulosa/química , Celulosa/metabolismo , Endo-1,3(4)-beta-Glucanasa/antagonistas & inhibidores , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glucanos/química , Glucanos/metabolismo , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Calor , Concentración de Iones de Hidrógeno , Mutación , Filogenia , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Xilanos/química , Xilanos/metabolismoRESUMEN
A series of N-bromoacetylglycosylamines and bromoketone C-glycosides were synthesised from complex xyloglucan oligosaccharide (XyGO) scaffolds as specific active-site affinity labels for endo-xyloglucanases. Compounds based on XXXG (Xyl3 Glc4 ) and XLLG (Xyl3 Glc4 Gal2 ) oligosaccharides exhibited strikingly higher affinities and higher rates of irreversible inhibition than known cellobiosyl and new lactosyl disaccharide congeners when tested with endo-xyloglucanases from two distinct glycoside hydrolase (GH) families. Intact-protein mass spectrometry indicated that inactivation with XyGO derivatives generally resulted in a 1:1 labelling stoichiometry. Together, these results indicate that XyGO-based affinity reagents have significant potential as inhibitors and proteomic reagents for the identification and analysis of diverse xyloglucan-active enzymes in nature, to facilitate industrial enzyme applications.
Asunto(s)
Bacteroides/enzimología , Clostridium thermocellum/enzimología , Glicósido Hidrolasas/antagonistas & inhibidores , Oligosacáridos/química , Oligosacáridos/farmacología , Secuencia de Carbohidratos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucanos/metabolismo , Glicósido Hidrolasas/metabolismo , Datos de Secuencia Molecular , Especificidad por Sustrato , Xilanos/metabolismoRESUMEN
A series of alpha-cyclodextrin derivatives containing a 3, 4 or 5 membered ether-linked bridge between the 6A and 6D oxygen atoms, with and without a ketone, were prepared. The synthesis used perbenzylated alpha-cyclodextrin A,D-diol as a starting material upon which O-alkylation and further modifications led to the di-O-(buta-1,4-diyl), the di-O-(penta-1,5-diyl) and the di-O-(buta-2-on-1,4-diyl) derivatives, which were debenzylated, and compared with the previously reported di-O-(propa-2-on-1,3-diyl) derivative. Permethylated derivatives of the di-O-(propa-2-on-1,3-diyl) and the buta-1,4-diyl derivatives were also made. The 6A,6D-di-O-(propa-2-on-1,3-diyl)-6C,6F-di-O-methyl and di-O-pivaloyl derivatives were also prepared. The new compounds were analysed for catalysis of the oxidation of amines and alcohols.