title: Structural analysis of glucuronoxylan-specific Xyn30D and its attached CBM35 domain gives insights into the role of modularity in specificity.
abstract: Glucuronoxylanase Xyn30D is a modular enzyme containing a family 30 glycoside hydrolase catalytic domain and an attached carbohydrate binding module of the CBM35 family. We present here the three-dimensional structure of the full-length Xyn30D at 2.4 Å resolution. The catalytic domain folds into an (α/β)8 barrel with an associated β-structure, whereas the attached CBM35 displays a jellyroll β-sandwich including two calcium ions. Although both domains fold in an independent manner, the linker region makes polar interactions with the catalytic domain, allowing a moderate flexibility. The ancillary Xyn30D-CBM35 domain has been expressed and crystallized, and its binding abilities have been investigated by soaking experiments. Only glucuronic acid-containing ligands produced complexes, and their structures have been solved. A calcium-dependent glucuronic acid binding site shows distinctive structural features as compared with other uronic acid-specific CBM35s, because the presence of two aromatic residues delineates a wider pocket. The nonconserved Glu(129) makes a bidentate link to calcium and defines region E, previously identified as specificity hot spot. The molecular surface of Xyn30D-CBM35 shows a unique stretch of negative charge distribution extending from its binding pocket that might indicate some oriented interaction with its target substrate. The binding ability of Xyn30D-CBM35 to different xylans was analyzed by affinity gel electrophoresis. Some binding was observed with rye glucuronoarabinoxylan in presence of calcium chelating EDTA, which would indicate that Xyn30D-CBM35 might establish interaction to other components of xylan, such as arabinose decorations of glucuronoarabinoxylan. A role in depolymerization of highly substituted chemically complex xylans is proposed.
authors.affiliation: From the Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, 28006 Madrid, Spain and.
authors.forename: M Angela
authors.initials: MA
authors.lastname: Sainz-Polo
authors.affiliation: the Departamento de Microbiología, Facultad de Biología. Universidad de Barcelona. 08028 Barcelona, Spain.
authors.forename: Susana Valeria
authors.initials: SV
authors.lastname: Valenzuela
authors.affiliation: From the Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, 28006 Madrid, Spain and.
authors.forename: Beatriz
authors.initials: B
authors.lastname: González
authors.affiliation: the Departamento de Microbiología, Facultad de Biología. Universidad de Barcelona. 08028 Barcelona, Spain.
authors.forename: F I Javier
authors.initials: FI
authors.lastname: Pastor
authors.affiliation: From the Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, 28006 Madrid, Spain and xjulia@iqfr.csic.es.
authors.forename: Julia
authors.initials: J
authors.lastname: Sanz-Aparicio
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D018533:Biomass; D020134:Catalytic Domain; D002473:Cell Wall / Q000201:enzymology; D018360:Crystallography, X-Ray; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology*; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D012434:Secale / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014934:Wood; D014990:Xylans / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D014990:Xylans; D004492:Edetic Acid; D014995:Xylosidases; C068045:glucuronoxylan xylanohydrolase
keywords: CBM35; Carbohydrate-binding Protein; Enzyme Mechanism; Enzyme Structure; GH30; Glucurooxylanase; Glycoside Hydrolase; Plant Cell Wall; X-ray crystallography