title: The Mechanism by Which Arabinoxylanases Can Recognize Highly Decorated Xylans.
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 O3to 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.
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Aurore
authors.initials: A
authors.lastname: Labourel
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Lucy I
authors.initials: LI
authors.lastname: Crouch
authors.affiliation: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal, NZYTech Genes & Enzymes, 1649-038 Lisboa, Portugal.
authors.forename: Joana L A
authors.initials: JL
authors.lastname: Brás
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Adam
authors.initials: A
authors.lastname: Jackson
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Artur
authors.initials: A
authors.lastname: Rogowski
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Joseph
authors.initials: J
authors.lastname: Gray
authors.affiliation: the Eastern Regional Research Center, United States Department of Agriculture-Agricultural Research Service, Wyndmoor, Pennsylvania 19038.
authors.forename: Madhav P
authors.initials: MP
authors.lastname: Yadav
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR7857 CNRS, Aix-Marseille University, F-13288 Marseille, France, USC1408 Architecture et Fonction des Macromolécules Biologiques, INRA, F-13288 Marseille, France, and the Department of Biological Sciences, King Abdulaziz University, Jedda 21589, Saudi Arabia.
authors.forename: Bernard
authors.initials: B
authors.lastname: Henrissat
authors.affiliation: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal, NZYTech Genes & Enzymes, 1649-038 Lisboa, Portugal.
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
authors.affiliation: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal, shabir@fmv.ulosboa.pt.
authors.forename: Shabir
authors.initials: S
authors.lastname: Najmudin
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom, arnaud.basle@ncl.uk.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Baslé
authors.affiliation: From the Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom, Fiona.cuskin@newcastle.ac.uk.
authors.forename: Fiona
authors.initials: F
authors.lastname: Cuskin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D018360:Crystallography, X-Ray; D000072417:Protein Domains; D014990:Xylans / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D014990:Xylans; C085118:arabinoxylan; D014995:Xylosidases; C110296:arabinoxylanase
keywords: cellulosome; crystallography; enzyme kinetics; enzyme mechanism; glycoside hydrolase