Structural Basis for Xyloglucan Specificity and α-d-Xylp(1 → 6)-d-Glcp Recognition at the −1 Subsite within the GH5 Family

Abstract

GH5 is one of the largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the crystal structures of two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 XEGs from parental cellulases and a new mode of action within the GH5 family related to structural adaptations in the -1 subsite. The oligosaccharide found in the XEG5A complex, permitted the mapping, for the first time, of the positive subsites of a GH5 XEG, revealing the importance of the pocket-like topology of the +1 subsite in conferring the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the negative subsites, completing the subsite mapping of GH5 XEGs at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl side chain at this position. The stereochemical compatibility of the -1 subsite with a xylosyl moiety was also reported for other structurally nonrelated XEGs belonging to the GH74 family, indicating it to be an essential attribute for this mode of action.

Structure-Function Analysis of a Mixed-linkage β-Glucanase/Xyloglucanase from the Key Ruminal Bacteroidetes Prevotella bryantii B14

“…Of the GH5 endo-xyloglucanases characterized to date, all cleave the dicot xyloglucan polysaccharide (exemplified by Tamarindus indica xyloglucan) at the unbranched backbone glucosyl unit (Fig. 1) to generate oligosaccharides based on a Glc 4 backbone (16,17,64). This cleavage pattern is also typical for GH7, GH9, GH12, and GH16 members, with known exceptions of certain GH44 and GH74 members (16, 18, 66 -71).…”

“…Hence, we compared PbGH5A to other well characterized GH5_4 enzymes as follows: P. pabuli XG5 (PpXG5, PDB code 2JEQ) (16); B. ovatus (BoGH5, PDB code 3ZMR) (17); Bacillus halodurans GH5 (BhGH5, PDB code 4V2X) (63); and Xeg5A (PDB code 4W88) and Xeg5B (PDB code 4W8B) (64). These were chosen as they have been subjected to detailed structure-function characterization and have been specifically tested for both mixed-linkage endo-glucanase and endo-xyloglucanase activities.…”

Crystal structure determination ofPseudomonas stutzeriA1501 endoglucanase Cel5A: the search for a molecular basis for glycosynthesis in GH5_5 enzymes

“…3a). The presence of a Tris molecule in the active site of GH5 enzymes has been reported in RBcel1 (Delsaute et al, 2013), the -mannanase from Trichoderma reesei (Sabini et al, 2000) and the xyloglucanase XEG5A (dos Santos et al, 2015).…”

Explorando as relações entre estrutura e função das hidrolases de glicosídeos das famílias 9, 48 e 74

“…A gram-positive bacterium, Bacillus licheniformis is a facultative anaerobe, which is widely distributed in the environment [29]. B. licheniformis has a number of important biotechnological, agricultural and industrial applications [30,31]. It is used for production of antibiotics, chemicals [30] and some of the industrially important hydrolytic enzymes including penicillinase, pentosanases, proteases, α-amylases, glucoamylase, pectinases and several cellulase enzymes [32][33][34].…”

“…B. licheniformis has a number of important biotechnological, agricultural and industrial applications [30,31]. It is used for production of antibiotics, chemicals [30] and some of the industrially important hydrolytic enzymes including penicillinase, pentosanases, proteases, α-amylases, glucoamylase, pectinases and several cellulase enzymes [32][33][34]. In this work, we report the heterologous expression, purification and biochemical characterization of BlCel48 from Bacillus licheniformis (ATCC 14580).…”