Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

DOI: 10.1073/pnas.1601558113

Authors: Immacolata Venditto (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda; Newcastle Universit) , Ana S. Luis (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda; Newcastle University) , Maja Rydahl (University of Copenhagen) , Julia Schückel (University of Copenhagen) , Vânia O. Fernandes (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda; NZYTech Genes & Enzymes) , Silvia Vidal-Melgosa (University of Copenhagen) , Pedro Bule (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda) , Arun Goyal (Indian Institute of Technology Guwahati) , Virginia M. R. Pires (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda) , Catarina G. Dourado (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda) , Luís M. A. Ferreira (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda; NZYTech Genes & Enzymes) , Pedro M. Coutinho (UMR 7857 CNRS, Aix-Marseille University) , Bernard Henrissat (UMR 7857 CNRS, Aix-Marseille University; Institut National de la Recherche Agronomique; King Abdulaziz University) , J. Paul Knox (University of Leeds) , Arnaud Baslé (Newcastle University) , Shabir Najmudin (Universidade Técnica de Lisboa) , Harry J. Gilbert (Newcastle University) , William G. T. Willats (University of Copenhagen) , Carlos M. G. A. Fontes (Universidade de Lisboa, Pólo Universitário do Alto da Ajuda; NZYTech Genes & Enzymes)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 113 , PAGES 7136 - 7141

State: Published (Approved)
Published: June 2016
Diamond Proposal Number(s): 9948

Abstract: 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 CBMfamilies that targeted beta-glucans, beta-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 beta-glucans and beta-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.

Journal Keywords: carbohydrate-binding modules; protein-carbohydrate interactions; carbohydrate active enZYmes; cellulosomes

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I02-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography

Other Facilities: European Synchrotron Radiation Facility (Grenoble, France) and Soleil (Saint-Aubin, France)

Added On: 15/08/2016 11:01

Discipline Tags:

Biotechnology Biochemistry Catalysis Chemistry Structural biology Engineering & Technology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)