title: Unique contribution of the cell wall-binding endoglucanase G to the cellulolytic complex in Clostridium cellulovorans.
abstract: The cellulosomes produced by Clostridium cellulovorans are organized by the specific interactions between the cohesins in the scaffolding proteins and the dockerins of the catalytic components. Using a cohesin biomarker, we identified a cellulosomal enzyme which belongs to the glycosyl hydrolase family 5 and has a domain of unknown function 291 (DUF291) with functions similar to those of the surface layer homology domain in C. cellulovorans. The purified endoglucanase G (EngG) had the highest synergistic degree with exoglucanase (ExgS) in the hydrolysis of crystalline cellulose (EngG/ExgS ratio = 3:1; 1.71-fold). To measure the binding affinity of the dockerins in EngG for the cohesins of the main scaffolding protein, a competitive enzyme-linked interaction assay was performed. Competitors, such as ExgS, reduced the percentage of EngG that were bound to the cohesins to less than 20%; the results demonstrated that the cohesins prefer to bind to the common cellulosomal enzymes rather than to EngG. Additionally, in surface plasmon resonance analysis, the dockerin in EngG had a relatively weak affinity (30- to 123-fold) for cohesins compared with the other cellulosomal enzymes. In the cell wall affinity assay, EngG anchored to the cell surfaces of C. cellulovorans using its DUF291 domain. Immunofluorescence microscopy confirmed the cell surface display of the EngG complex. These results indicated that in C. cellulovorans, EngG assemble into both the cellulolytic complex and the cell wall complex to aid in the hydrolysis of cellulose substrates.
authors.affiliation: School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea.
authors.forename: Sang Duck
authors.initials: SD
authors.lastname: Jeon
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Su Jung
authors.initials: SJ
authors.lastname: Kim
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Park
authors.forename: Gi-Wook
authors.initials: GW
authors.lastname: Choi
authors.forename: Sung Ok
authors.initials: SO
authors.lastname: Han
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D046974:Clostridium cellulovorans / Q000201:enzymology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D020349:Surface Plasmon Resonance
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D002480:Cellulase