title: A bacterial GH6 cellobiohydrolase with a novel modular structure.
abstract: Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn31,2) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a Kmof 68.73 mg/ml and a Vmaxof 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
authors.forename: Liliana
authors.initials: L
authors.lastname: Cerda-Mejía
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
authors.forename: Susana Valeria
authors.initials: SV
authors.lastname: Valenzuela
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Frías
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain.
authors.forename: Pilar
authors.initials: P
authors.lastname: Diaz
authors.affiliation: Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, Av. Diagonal 643, 08028, Barcelona, Spain. fpastor@ub.edu.
authors.forename: F I Javier
authors.initials: FI
authors.lastname: Pastor
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020134:Catalytic Domain; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D056507:Paenibacillus / Q000201:enzymology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Exoglucanase; Modular GH6; Paenibacillus