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The mechanism by which arabinoxylanases can recognise highly decorated xylans

DOI: 10.1074/jbc.M116.743948 DOI Help

Authors: Aurore Labourel (Institute for Cell and Molecular Biosciences, Newcastle University) , Lucy I. Crouch (Institute for Cell and Molecular Biosciences, Newcastle University) , Joana L. A. Brás (CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa; NZYTech Genes & Enzymes) , Adam Jackson (Institute for Cell and Molecular Biosciences, Newcastle University) , Artur Rogowski (Institute for Cell and Molecular Biosciences, Newcastle University) , Joseph Gray (Institute for Cell and Molecular Biosciences, Newcastle University) , Madhav P. Yadav (‖Eastern Regional Research Center, United States Department of Agriculture-Agricultural Research Service) , Bernard Henrissat (Architecture et Fonction des Macromolécules Biologiques; ; Department of Biological Sciences, King Abdulaziz University) , Carlos M. G. A. Fontes (CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa; ; NZYTech Genes & Enzymes) , Harry J. Gilbert (Institute for Cell and Molecular Biosciences, Newcastle University) , Shabir Najmudin (CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa) , Arnaud Basle (Institute for Cell and Molecular Biosciences, Newcastle University) , Fiona Cuskin (Institute for Cell and Molecular Biosciences, Newcastle University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry

State: Published (Approved)
Published: August 2016
Diamond Proposal Number(s): 1960 , 7854 , 9948

Open Access Open Access

Abstract: The enzymatic degradation of plant cell walls is an important biological process of increasing environmental and industrial significance. Xylan, a major component of the plant cell wall, consists of a backbone of β-1,4-xylose (Xylp) units that are often decorated with arabinofuranose (Araf) side chains. A large penta-modular enzyme, CtXyl5A, was shown previously to specifically target arabinoxylans. The mechanism of substrate recognition displayed by the enzyme, however, remains unclear. Here we report the crystal structure of the arabinoxylanase and the enzyme in complex with ligands. The data showed that four of the protein modules adopt a rigid structure, which stabilizes the catalytic domain. The C-terminal non-catalytic carbohydrate binding module could not be observed in the crystal structure, suggesting positional flexibility. The structure of the enzyme in complex with Xylp-β-1,4-Xylp-β-1,4-Xylp-[α-1,3-Araf]-β-1,4-Xylp showed that the Araf decoration linked O3 to the xylose in the active site is located in the pocket (−2* subsite) that abuts onto the catalytic center. The −2* subsite can also bind to Xylp and Arap, explaining why the enzyme can utilize xylose and arabinose as specificity determinants. Alanine substitution of Glu68, Tyr92, or Asn139, which interact with arabinose and xylose side chains at the −2* subsite, abrogates catalytic activity. Distal to the active site, the xylan backbone makes limited apolar contacts with the enzyme, and the hydroxyls are solvent-exposed. This explains why CtXyl5A is capable of hydrolyzing xylans that are extensively decorated and that are recalcitrant to classic endo-xylanase attack.

Journal Keywords: cellulosome; crystallography; enzyme kinetics; enzyme mechanism; glycoside hydrolase

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I02-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I24-Microfocus Macromolecular Crystallography

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