Unveiling the Molecular Mechanism of a Novel Gh43 Exo-Oligoxylanase

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1st EuroCarb Young Researcher (ECYR) Workshop

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Mini symposium: Glycobiology at the LUMC and beyond

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Unveiling the Molecular Mechanism of a Novel Gh43 Exo-Oligoxylanase

Mariana Morais 1,2 , Joan Coines 2 , Mariane Domingues 1 , Renan Pirolla 1 , Carme Rovira 2, 3, Mario Murakami 1

1 Brazilian Bioethanol Science and Technology Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, Brazil, 2 Departament de Química Inorgànica i Orgànica & Institut de Química Teórica i Computacional, Universitat de Barcelona, Barcelona, Spain, 3 Institució Catalana de Recerca i Estudis Avançats , Barcelona, Spain

Glycoside hydrolases from family 43 (GH43) act on the degradation of key polysaccharides that constitute the hemicellulose such as xylan and arabinans and have a huge functional and structural diversity. Despite its strong correlation with plant cell wall deconstruction, being ubiquitous in plant biomass maceration specialized organisms, the catalytic mechanisms of family 43 GHs are still poorly understood. In this work, we characterized a xylose-releasing exo-oligoxylanase from Xanthomonas axonopodis pv. citri (Xac), the causative agent of citrus canker, by a multidisciplinary approach based on experimental and computational methods, including mass spectrometry for kinetics determination, X-ray crystallography and molecular dynamics simulations. The enzyme efficiently hydrolyzes xylooligosaccharides with preference for longer substrates and shows minor activity on polymeric xylan. As observed in other GH43 with different functions, the Xac exo-oligoxylanase activity is boosted by the presence of calcium, which decreases the Michaelis constant. To reveal the molecular basis of the enzyme’s activity, its crystal structure was solved, in the presence of calcium, without sugar ligand and with its product (xylose, X1) and substrate (xylotriose, X3). The Michaelis complex structure shows that the sugar at the -1 subsite exhibits a distorted conformation. Molecular dynamics simulations results reveal putative catalytic itineraries that are consistent with the conformational free energy landscape of β-xylose computed by ab initio metadynamics [1]. Altogether, our results expand our knowledge about this enigmatic GH family, revealing mechanistic details by a crosstalk between different methodologies.

Iglesias-Fernández, J.; Raich, L.; Ardèvol, A.; Rovira, C. The complete conformational free energy landscape of β-xylose reveals a two-fold catalytic itinerary for β-xylanases. Chem. Sci. 6, 1167-1177.

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