Structural and functional characterization of a ruminal β-glycosidase defines a novel subfamily of glycosyl hydrolase family 3 with permuted domain topology

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  • Mercedes Ramirez-Escudero
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Mercedes V. del Pozo
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Julia Marin-Navarro
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Beatriz Gonzalez
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Peter Golyshin
  • Julia Polaina
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Manuel Ferrer
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
  • Julia Sanz-Aparico
    Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC), Madrid
Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as it exemplified by glycosyl hydrolase (GH) family-3 proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose are among the preferred substrates, the large polysaccharide lichenan being also hydrolysed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identifying its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the broad substrate specificity. A hydrophobic platform defined by residues Trp711 and Trp106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidences that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials
Original languageEnglish
JournalJournal of Biological Chemistry
DOIs
StateE-pub ahead of print - 27 Sep 2016
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