title: Biochemical characterization and crystal structure of endoglucanase Cel5A from the hyperthermophilic Thermotoga maritima.
abstract: Tm_Cel5A, which belongs to family 5 of the glycoside hydrolases, is an extremely stable enzyme among the endo-acting glycosidases present in the hyperthermophilic organism Thermotoga maritima. Members of GH5 family shows a common (β/α)(8) TIM-barrel fold in which the catalytic acid/base and nucleophile are located on strands β-4 and β-7 of the barrel fold. Thermally resistant cellulases are desirable for lignocellulosic biofuels production and the Tm_Cel5A is an excellent candidate for use in the degradation of polysaccharides present on biomass. This paper describes two Tm_Cel5A structures (crystal forms I and II) solved at 2.20 and 1.85Å resolution, respectively. Our analyses of the Tm_Cel5A structure and comparison to a mesophilic GH5 provides a basis for the thermostability associated with Tm_Cel5A. Furthermore, both crystal forms of Tm_Cel5A possess a cadmium (Cd(2+)) ion bound between the two catalytic residues. Activity assays of Tm_Cel5A confirmed a strong inhibition effect in the presence of Cd(2+) metal ions demonstrating competition with the natural substrate for the active site. Based on the structural information we have obtained for Tm_Cel5A, protein bioengineering can be used to potentially increase the thermostability of mesophilic cellulase enzymes.
authors.affiliation: Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
authors.forename: Jose H
authors.initials: JH
authors.lastname: Pereira
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Chen
authors.forename: Ryan P
authors.initials: RP
authors.lastname: McAndrew
authors.forename: Rajat
authors.initials: R
authors.lastname: Sapra
authors.forename: Swapnil R
authors.initials: SR
authors.lastname: Chhabra
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Sale
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D055550:Protein Stability; D017433:Protein Structure, Secondary; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; C407024:endoglucanase Cel5A; D002480:Cellulase