title: Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus.
abstract: Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A, and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family, and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the -3 to -1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic "clamp", potentially interacting with sugars at the +1 and +2 subsites. The enzyme's active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, 1432 NORWAY.
authors.forename: A E
authors.initials: AE
authors.lastname: Naas
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, 1432 NORWAY.
authors.forename: A K
authors.initials: AK
authors.lastname: MacKenzie
authors.affiliation: Department of Medical Biochemistry, Institute for Clinical Medicine, University Of Oslo, PO Box 4950, Nydalen, N-0424, Oslo, NORWAY.|Department of Microbiology, Clinic for Diagnostics and Intervention, Oslo University Hospital, Rikshospitalet, PO Box 4950, Nydalen, N-0424, Oslo, NORWAY.
authors.forename: B
authors.initials: B
authors.lastname: Dalhus
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, 1432 NORWAY.
authors.forename: V G H
authors.initials: VGH
authors.lastname: Eijsink
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, 1432 NORWAY.
authors.forename: P B
authors.initials: PB
authors.lastname: Pope
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D041963:Bacteroidetes / Q000201:enzymology*; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry*; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002482:Cellulose; D002480:Cellulase