Structural basis of exo-β-mannanase activity in the GH2 family

Abstract

The classical microbial strategy for depolymerization of β-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-β-mannanases and β-mannosidases. In this work, we describe the first exo-β-mannanase from the GH2 family, isolated from pv. (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and β-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 β-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 β-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 β-mannosidases, Gly and Gly, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-β-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-β-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila

“…In addition, we found the following structural homologs for the β-sandwich domain using the same server: Supplementary Fig. 4d-f) 70 . SleM is a peptidoglycan lysin, composed of an N-terminal catalytic domain similar to the GH25 family lysozymes and a Cterminal FnIII domain.…”

Structure‐guided design combined with evolutionary diversity led to the discovery of the xylose‐releasing exo‐xylanase activity in the glycoside hydrolase family 43

“…The exo‐β‐1,4‐xylanase activity has been attributed to the presence of a shortened loop that distinguishes BlXynB from other GH43_11 and GH43_12 members by the loop length, composition and orientation, generating extra positive subsites in the BlXynB active site. A similar mechanism to BlXynB was recently unveiled for a GH2 exo‐mannanase, which is able to cleave both manno‐oligosaccharides and polymeric mannans, which resulted from the removal of physical barriers from the positive‐subsite region of an ancestral β‐mannosidase (Domingues et al, ).…”

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites

“…The fungal glycoside hydrolase GH18 is mainly responsible for remodeling and recovery of the fungal cell wall and other cell wall degrading enzymes [50]. The fungal glycoside hydrolases GH2 can specifically hydrolyze the β-glycosidic bond between D-glucuronidose and aglycon, and has important applications in the diagnosis and drug development of metabolic diseases [51]. We hypothesize that the expansion of GH18 family and GH2 family in C. arbuscula genome may contribute to help the host to resist the attack of other fungi.…”