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

• Published in: The Journal of biological chemistry Vol. 293; no. 35; pp. 13636 - 13649
• Main Authors: Domingues, Mariane Noronha, Souza, Flavio Henrique Moreira, Vieira, Plínio Salmazo, de Morais, Mariana Abrahão Bueno, Zanphorlin, Letícia Maria, dos Santos, Camila Ramos, Pirolla, Renan Augusto Siqueira, Honorato, Rodrigo Vargas, de Oliveira, Paulo Sergio Lopes, Gozzo, Fabio Cesar, Murakami, Mário Tyago
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31-08-2018; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; beta-Mannosidase - chemistry; beta-Mannosidase - metabolism; Catalytic Domain; Crystallography, X-Ray; enzyme; enzyme catalysis; Enzymology; exo-β-mannanase; GH2 family; glycoside hydrolase; Hydrolysis; Kinetics; Mannans - metabolism; Mannose - metabolism; Models, Molecular; molecular mechanism; Protein Conformation; protein structure; Scattering, Small Angle; Sequence Alignment; site-directed mutagenesis; Substrate Specificity; X-Ray Diffraction; Xanthomonas - chemistry; Xanthomonas - enzymology; Xanthomonas - metabolism; Xanthomonas axonopodis pv. citri; enzyme; protein structure; site-directed mutagenesis; glycoside hydrolase; molecular mechanism; Xanthomonas axonopodis pv. citri; enzyme catalysis; protein conformation; GH2 family; exo-β-mannanase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA118.002374

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.