title: Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome.
abstract: The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan.
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Hirano
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nihei
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hasegawa
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haruki
authors.affiliation: Department of Chemical Biology and Applied Chemistry, College of Engineering, Nihon University, Koriyama, Fukushima, Japan Japan Science and Technology Agency, PRESTO, Kawaguchi, Saitama, Japan nhirano@chem.ce.nihon-u.ac.jp.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hirano
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D044602:Cellulases / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D044603:Cellulosomes / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D003460:Crystallization
chemical_list: D001426:Bacterial Proteins; D002482:Cellulose; D044602:Cellulases