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 Xanthomonas axonopodis pv. citri (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, Gly439 and Gly556, 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.
Highlights
The classical microbial strategy for depolymerization of -mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4--mannanases and -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 -mannosidases, generating a more open and accessible active site with additional productive positive subsites
The only other mechanistically characterized mannose-releasing exo--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
Summary
XacMan2A is an exo--mannanase from the GH2 family The X. axonopodis pv. citri GH2 enzyme AAM37920.1) ORF comprises 2,691 bp encoding an 886-amino acid protein with a molecular mass of 99.48 kDa. The recombinant XacMan2A was successfully overexpressed in BL21(DE3) Escherichia coli cells, and the purification procedure yielded 2.5 mg of pure and homogenous enzyme per liter of culture, despite its large size and structural complexity. CD analysis indicated a proper folded conformation with a melting temperature of 47.6 °C (Fig. 1A), which is similar to that observed in other CAZyme isolated from the same bacterium [21]. XacMan2A thermal unfolding followed the canonical two-state model with a single transition (Fig. 1A), despite the five-domain organization inferred by sequence analysis and further confirmed by X-ray crystallography. Activity assays with synthetic substrates revealed that XacMan2A is only active on p-nitrophenyl--D-mannopyranoside (pNP--Man) exhibiting optimum pH and temperature of 5.5 and 40 °C, respectively, with a moderate thermotolerance (Fig. 1, B–D). XacMan2A was able to cleave mannose-based polysaccharides, showing high activity against
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