title: Functional and modular analyses of diverse endoglucanases from Ruminococcus albus 8, a specialist plant cell wall degrading bacterium.
abstract: Ruminococcus albus 8 is a specialist plant cell wall degrading ruminal bacterium capable of utilizing hemicellulose and cellulose. Cellulose degradation requires a suite of enzymes including endoglucanases, exoglucanases, and β-glucosidases. The enzymes employed by R. albus 8 in degrading cellulose are yet to be completely elucidated. Through bioinformatic analysis of a draft genome sequence of R. albus 8, seventeen putatively cellulolytic genes were identified. The genes were heterologously expressed in E. coli, and purified to near homogeneity. On biochemical analysis with cellulosic substrates, seven of the gene products (Ra0185, Ra0259, Ra0325, Ra0903, Ra1831, Ra2461, and Ra2535) were identified as endoglucanases, releasing predominantly cellobiose and cellotriose. Each of the R. albus 8 endoglucanases, except for Ra0259 and Ra0325, bound to the model crystalline cellulose Avicel, confirming functional carbohydrate binding modules (CBMs). The polypeptides for Ra1831 and Ra2535 were found to contain distantly related homologs of CBM65. Mutational analysis of residues within the CBM65 of Ra1831 identified key residues required for binding. Phylogenetic analysis of the endoglucanases revealed three distinct subfamilies of glycoside hydrolase family 5 (GH5). Our results demonstrate that this fibrolytic bacterium uses diverse GH5 catalytic domains appended with different CBMs, including novel forms of CBM65, to degrade cellulose.
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Iakiviak
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Saravanan
authors.initials: S
authors.lastname: Devendran
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Skorupski
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Young Hwan
authors.initials: YH
authors.lastname: Moon
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Roderick I
authors.initials: RI
authors.lastname: Mackie
authors.affiliation: Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.|Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Isaac
authors.initials: I
authors.lastname: Cann
mesh_terms: D002473:Cell Wall / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D004252:DNA Mutational Analysis; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010756:Phosphoric Acids / Q000737:chemistry; D059828:Plant Cells / Q000378:metabolism*; D000072417:Protein Domains; D045857:Ruminococcus / Q000201:enzymology*; D012995:Solubility; D013379:Substrate Specificity
chemical_list: D010756:Phosphoric Acids; D002482:Cellulose; C030242:phosphoric acid; D002480:Cellulase