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Characterization of a Hexameric Exo-Acting GH51 α-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis

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Abstract

α-l-Arabinofuranosidases (α-l-Abfases, EC 3.2.1.55) display a broad specificity against distinct glycosyl moieties in branched hemicellulose and recent studies have demonstrated their synergistic use with cellulases and xylanases for biotechnological processes involving plant biomass degradation. In this study, we examined the structural organization of the arabinofuranosidase (GH51 family) from the mesophilic Bacillus subtilis (AbfA) and its implications on function and stability. The recombinant AbfA showed to be active over a broad temperature range with the maximum activity between 35 and 50 °C, which is desirable for industrial applications. Functional studies demonstrated that AbfA preferentially cleaves debranched or linear arabinan and is an exo-acting enzyme producing arabinose from arabinoheptaose. The enzyme has a canonical circular dichroism spectrum of α/β proteins and exhibits a hexameric quaternary structure in solution, as expected for GH51 members. Thermal denaturation experiments indicated a melting temperature of 53.5 °C, which is in agreement with the temperature–activity curves. The mechanisms associated with the unfolding process were investigated through molecular dynamics simulations evidencing an important contribution of the quaternary arrangement in the stabilization of the β-sandwich accessory domain and other regions involved in the formation of the catalytic interface of hexameric Abfases belonging to GH51 family.

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Acknowledgments

This study was financially supported by Grants from the Brazilian agencies FAPESP (2008/58037-9, 10/51890-8) and CNPQ (133394/2011-5, 475022/2011-4, 310177/2011-1 and 2011/17658-3). ZBH, LCO and TMA received fellowship from FAPESP (2011/14200-6, 2011/13242-7, 2010/11499-1, respectively). JC receives fellowship from CNPq (140420/2009-6). We gratefully acknowledge the provision of time on the SAXS2 beamline at LNLS and spectroscopy facilities at LNBio. Computational resources were supplied by NCC/GridUNESP from UNESP and CENAPAD-SP (Project UNICAMP/FINEP-MCT). We are grateful to Professor Richard John Ward for technical assistance and text correction.

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Correspondence to Roberto Ruller.

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Zaira B. Hoffmam and Leandro C. Oliveira have contributed equally to this work.

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Hoffmam, Z.B., Oliveira, L.C., Cota, J. et al. Characterization of a Hexameric Exo-Acting GH51 α-l-Arabinofuranosidase from the Mesophilic Bacillus subtilis . Mol Biotechnol 55, 260–267 (2013). https://doi.org/10.1007/s12033-013-9677-1

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