Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition.
The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
Immacolata
I
Venditto
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
Ana S
AS
Luis
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;
Maja
M
Rydahl
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;
Julia
J
Schückel
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; NZYTech Genes & Enzymes, Campus do Lumiar, 1649-038 Lisbon, Portugal;
Vânia O
VO
Fernandes
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark;
Silvia
S
Vidal-Melgosa
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal;
Pedro
P
Bule
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam, India;
Arun
A
Goyal
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal;
Virginia M R
VM
Pires
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal;
Catarina G
CG
Dourado
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; NZYTech Genes & Enzymes, Campus do Lumiar, 1649-038 Lisbon, Portugal;
Luís M A
LM
Ferreira
Architecture et Fonction des Macromolécules Biologiques, UMR 7857 CNRS, Aix-Marseille University, F-13288 Marseille, France;
Pedro M
PM
Coutinho
Architecture et Fonction des Macromolécules Biologiques, UMR 7857 CNRS, Aix-Marseille University, F-13288 Marseille, France; Institut National de la Recherche Agronomique, USC 1408 Architecture et Fonction des Macromolécules Biologiques, F-13288 Marseille, France, Department of Biological Sciences, King Abdulaziz University, Jeddah, Saudi Arabia;
Bernard
B
Henrissat
Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
J Paul
JP
Knox
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom;
Arnaud
A
Baslé
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal;
Shabir
S
Najmudin
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; cafontes@fmv.ulisboa.pt willats@plen.ku.dk harry.gilbert@ncl.ac.uk.
Harry J
HJ
Gilbert
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark; cafontes@fmv.ulisboa.pt willats@plen.ku.dk harry.gilbert@ncl.ac.uk.
William G T
WG
Willats
Interdisciplinary Centre of Research in Animal Health, Faculdade de Medicina Veterinária, Universidade de Lisboa, Pólo Universitário do Alto da Ajuda, 1300-477 Lisbon, Portugal; NZYTech Genes & Enzymes, Campus do Lumiar, 1649-038 Lisbon, Portugal; cafontes@fmv.ulisboa.pt willats@plen.ku.dk harry.gilbert@ncl.ac.uk.
Carlos M G A
CM
Fontes
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044603:Cellulosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D045857:Ruminococcus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011134:Polysaccharides
carbohydrate active enZYmes; carbohydrate-binding modules; cellulosomes; protein-carbohydrate interactions