A rhamnogalacturonan lyase in the Clostridium cellulolyticum cellulosome.
Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wall-degrading activities named cellulosomes. Most of the genes encoding cellulosomal components are located in a large gene cluster: cipC-cel48F-cel8C-cel9G-cel9E-orfX-cel9H-cel9J-man5K-cel9M. Downstream of the cel9M gene, a new open reading frame was discovered and named rgl11Y. Amino acid sequence analysis indicates that this gene encodes a multidomain pectinase, Rgl11Y, containing an N-terminal signal sequence, a catalytic domain belonging to family 11 of the polysaccharide lyases, and a C-terminal dockerin domain. The present report describes the biochemical characterization of a recombinant form of Rgl11Y. Rgl11Y cleaves the alpha-L-Rhap-(1-->4)-alpha-D-GalpA glycosidic bond in the backbone of rhamnogalacturonan I (RGI) via a beta-elimination mechanism. Its specific activity on potato pectic galactan and rhamnogalacturonan was found to be 28 and 3.6 IU/mg, respectively, indicating that Rgl11Y requires galactan decoration of the RGI backbone. The optimal pH of Rgl11Y is 8.5 and calcium is required for its activity. Rgl11Y was shown to be incorporated in the C. cellulolyticum cellulosome through a typical cohesin-dockerin interaction. Rgl11Y from C. cellulolyticum is the first cellulosomal rhamnogalacturonase characterized.
Pagès
Sandrine
S
Laboratoire de Bioénergétique et Ingénierie des Protéines, IBSM, Centre National de la Recherche Scientifique, Marseille, France. pages@ibsm.cnrs-mrs.fr
Valette
Odile
O
Abdou
Laetitia
L
Bélaïch
Anne
A
Bélaïch
Jean-Pierre
JP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010368:Pectins / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010368:Pectins; D011133:Polysaccharide-Lyases