Structural basis of exo-β-mannanase activity in the GH2 family.
| Autor: | Domingues MN; From the Brazilian Bioethanol Science and Technology Laboratory and., Souza FHM; From the Brazilian Bioethanol Science and Technology Laboratory and., Vieira PS; From the Brazilian Bioethanol Science and Technology Laboratory and., de Morais MAB; From the Brazilian Bioethanol Science and Technology Laboratory and., Zanphorlin LM; From the Brazilian Bioethanol Science and Technology Laboratory and., Dos Santos CR; From the Brazilian Bioethanol Science and Technology Laboratory and., Pirolla RAS; From the Brazilian Bioethanol Science and Technology Laboratory and., Honorato RV; Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, São Paulo 13083-970, Brazil and., de Oliveira PSL; Brazilian Biosciences National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, São Paulo 13083-970, Brazil and., Gozzo FC; the Dalton Mass Spectrometry Laboratory, University of Campinas, Campinas, São Paulo 13083-970, Brazil., Murakami MT; From the Brazilian Bioethanol Science and Technology Laboratory and mario.murakami@ctbe.cnpem.br. |
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| Jazyk: | angličtina |
| Zdroj: | The Journal of biological chemistry [J Biol Chem] 2018 Aug 31; Vol. 293 (35), pp. 13636-13649. Date of Electronic Publication: 2018 Jul 11. |
| DOI: | 10.1074/jbc.RA118.002374 |
| Abstrakt: | 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, Gly 439 and Gly 556 , 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. (© 2018 Domingues et al.) |
| Databáze: | MEDLINE |
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