Structural analysis of glucuronoxylan-specific Xyn30D and its attached CBM35 domain gives insights into the role of modularity in specificity

J Biol Chem. 2014 Nov 7;289(45):31088-101. doi: 10.1074/jbc.M114.597732. Epub 2014 Sep 8.

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.

Keywords: CBM35; Carbohydrate-binding Protein; Enzyme Mechanism; Enzyme Structure; GH30; Glucurooxylanase; Glycoside Hydrolase; Plant Cell Wall; X-ray crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus subtilis / enzymology
  • Bacterial Proteins / chemistry*
  • Binding Sites
  • Biomass
  • Catalytic Domain
  • Cell Wall / enzymology
  • Crystallography, X-Ray
  • Edetic Acid / chemistry
  • Escherichia coli / metabolism
  • Molecular Sequence Data
  • Paenibacillus / enzymology*
  • Polysaccharides / chemistry
  • Protein Binding
  • Secale / chemistry
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Wood
  • Xylans / chemistry*
  • Xylosidases / chemistry*

Substances

  • Bacterial Proteins
  • Polysaccharides
  • Xylans
  • Edetic Acid
  • Xylosidases
  • glucuronoxylan xylanohydrolase

Associated data

  • PDB/4QAW
  • PDB/4QB1
  • PDB/4QB2
  • PDB/4QB6