title: An intermolecular disulfide bond is required for thermostability and thermoactivity of β-glycosidase from Thermococcus kodakarensis KOD1.
abstract: Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. β-glycosidase, Tkβgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkβgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the kcat/Km of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkβgly enzyme.
authors.affiliation: Department of Molecular Science and Engineering, National Taipei University of Technology, Taipei, Taiwan, kyhwa@ntut.edu.tw.
authors.forename: Kuo-Yuan
authors.initials: KY
authors.lastname: Hwa
authors.forename: Boopathi
authors.initials: B
authors.lastname: Subramani
authors.forename: San-Tai
authors.initials: ST
authors.lastname: Shen
authors.forename: Yu-May
authors.initials: YM
authors.lastname: Lee
mesh_terms: D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D004220:Disulfides; D006026:Glycoside Hydrolases