title: Purification and crystallographic studies of a putative carbohydrate-binding module from the Ruminococcus flavefaciens FD-1 endoglucanase Cel5A.
abstract: Ruminant herbivores meet their carbon and energy requirements from a symbiotic relationship with cellulosome-producing anaerobic bacteria that efficiently degrade plant cell-wall polysaccharides. The assembly of carbohydrate-active enzymes (CAZymes) into cellulosomes enhances protein stability and enzyme synergistic interactions. Cellulosomes comprise diverse CAZymes displaying a modular architecture in which a catalytic domain is connected, via linker sequences, to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs direct the appended catalytic modules to their target substrates, thus facilitating catalysis. The genome of the ruminal cellulolytic bacterium Ruminococcus flavefaciens strain FD-1 contains over 200 modular proteins containing the cellulosomal signature dockerin module. One of these is an endoglucanase Cel5A comprising two family 5 glycoside hydrolase catalytic modules (GH5) flanking an unclassified CBM (termed CBM-Rf2) and a C-terminal dockerin. This novel CBM-Rf2 has been purified and crystallized, and data from cacodylate-derivative crystals were processed to 1.02 and 1.29 Å resolution. The crystals belonged to the orthorhombic space group P212121. The CBM-Rf2 structure was solved by a single-wavelength anomalous dispersion experiment at the As edge.
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Ana José
authors.initials: AJ
authors.lastname: Pires
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Ribeiro
authors.affiliation: Synchrotron SOLEIL, L'Orme des Merisiers, Saint-Aubin, 91192 Gif-sur-Yvette, France.
authors.forename: Andrew
authors.initials: A
authors.lastname: Thompson
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Venditto
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Vânia O
authors.initials: VO
authors.lastname: Fernandes
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Bule
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Helena
authors.initials: H
authors.lastname: Santos
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Victor D
authors.initials: VD
authors.lastname: Alves
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Virginia
authors.initials: V
authors.lastname: Pires
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Luis M A
authors.initials: LM
authors.lastname: Ferreira
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.affiliation: CIISA, Faculdade de Medicina Veterinari, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal.
authors.forename: Shabir
authors.initials: S
authors.lastname: Najmudin
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D045857:Ruminococcus / Q000737:chemistry* / Q000201:enzymology; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C407024:endoglucanase Cel5A; D002480:Cellulase
keywords: Ruminococcus flavefaciens; carbohydrate-active enzymes; carbohydrate-binding module; cellulosomes; glycoside hydrolase