Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungusRasamsonia emersonii

Abstract

The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. -Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the -glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the -glucosidase from H. jecorina (HjCel3A) with the -glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 -glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures.

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations

“…Disease-associated mutations that are not localized in the catalytic domain might interfere with the structural integrity of the GBA2 protein, with protein-protein interactions, or protein association with membranes. Some members of other ␤-glucosidase families have been proposed to form dimers, which assemble as tetramers (22,23). Thus, GBA2 might also form oligomeric complexes.…”

Strategies for carbohydrate model building, refinement and validation

“…This was originally suggested by Stewart-Jones et al (2016), later proposed by and recently adopted by Gudmundsson et al (2016).…”

“…While ligand carbohydrates continue to be the focus of many biotechnological and biomedical studies, it would seem that the contribution of glycosylation to eukaryotic protein folding, stability and function is progressively taking the spotlight. This is already having implications: while the number of ligand sugars per structure will usually be within one to a couple of dozen at most, heavily glycosylated structures are becoming more frequent and can contain over 100 monosaccharides each (see, for example, Gudmundsson et al, 2016;Stewart-Jones et al, 2016), increasing the number of deposited monosaccharide models per year. Cryo-electron microscopy (cryo-EM), a structural technique that does not depend on the ordered packing of particles into crystals, is not vulnerable to the deleterious effects that external glycans may have (Pallesen et al, 2016), and thus is expected to contribute strongly to this trend in forthcoming years.…”

“…An aperiodic torsion restraint enforces a single torsional value, and by applying a set of torsion values to the ring bonds, it is possible to enforce one particular conformation. This has been shown to work well in a number of examples Gudmundsson et al, 2016;Agirre, Iglesias-Ferná ndez et al, 2015). LINK records can be created with Coot by choosing the 'Make link .…”

Sistemas mono-enzimáticos de desglicosilación de flavonoides (diglicosidasas) para su aplicación en procesos tecnológicos

“…El análisis filogenético de las proteínas fúngicas hipotéticas y de las β-glucosidasas cristalizadas con mayor similitud con αRβG II seleccionadas desde el análisis de BLAST (H. jecorina, Rasamsonia emersonii, Thermotoga neapolitana y Kluyveromyces marxianus) se agruparon dentro de la familia GH3 (Fig 34) El alineamiento de las secuencias de αRβG II con las proteínas GH3 caracterizadas estructuralmente permitió reconocer la presencia de residuos conservados. Los resultados de este trabajo refuerzan la hipótesis de que el subsitio -1 tiene una composición conservada (Karkehabadi et al, 2014;Suzuki et al, 2013;Gudmundsson et al, 2016;Yoshida et al, 2010;Pozzo et al, 2010), siendo Asp 245, Glu 475, Asp 62, Arg 125, Lys 158 y His 159 los residuos involucrados en los enlaces puentes de hidrogeno con glucosa (Fig. 35) Figura 35: Alineamiento de las secuencias de las glicosidasas cristalizadas pertenecientes a la familia GH 3 y αRβG II.…”