title: Dissecting structure-function-stability relationships of a thermostable GH5-CBM3 cellulase from Bacillus subtilis 168.
abstract: Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.
authors: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas SP, Brazil.
authors: Camila R
authors: CR
authors: Santos
authors: Joice H
authors: JH
authors: Paiva
authors: Maurício L
authors: ML
authors: Sforça
authors: Jorge L
authors: JL
authors: Neves
authors: Rodrigo Z
authors: RZ
authors: Navarro
authors: Júnio
authors: J
authors: Cota
authors: Patrícia K
authors: PK
authors: Akao
authors: Zaira B
authors: ZB
authors: Hoffmam
authors: Andréia N
authors: AN
authors: Meza
authors: Juliana H
authors: JH
authors: Smetana
authors: Maria L
authors: ML
authors: Nogueira
authors: Igor
authors: I
authors: Polikarpov
authors: José
authors: J
authors: Xavier-Neto
authors: Fábio M
authors: FM
authors: Squina
authors: Richard J
authors: RJ
authors: Ward
authors: Roberto
authors: R
authors: Ruller
authors: Ana C
authors: AC
authors: Zeri
authors: Mário T
authors: MT
authors: Murakami
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D044602:Cellulases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D006358:Hot Temperature; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008345:Manganese; D044602:Cellulases; D002118:Calcium