title: A single free cysteine residue and disulfide bond contribute to the thermostability of Aspergillus saitoi 1,2-alpha-mannosidase.
abstract: Aspergillus saitoi 1,2-alpha-mannosidase contains three conserved cysteine residues (Cys334, Cys363, and Cys443). We showed that Cys334 and Cys363 are involved in a disulfide bond, and that Cys443 contains a free thiol group. The cysteines were not essential for the activity analyzed by site-directed mutagenesis and kinetics. The substitution at each cysteine residue greatly destabilized the enzyme. The T(m) values of WT, C443A, C443G, C443S, and C443T were 55.8, 51.9, 50.2, 50.0, and 52.8 degrees C respectively. The specific activity of these mutants was almost equal to that of WT. Introducing Asp, Leu, Met, or Val at position 443 caused partial denaturation, although the enzymes had some activity. C443F, C443I, C443N, and C443Y were not secreted. These results suggest that the hydrophilic and large side chain causes the destabilization. Molecular modelling showed that the Cys443 residue is buried and surrounded by a hydrophobic environment. Cys334 and Cys363 form a disulfide bond, and Cys443 is involved in a hydrophobic interaction to stabilize the enzyme.
authors.affiliation: Laboratory of Molecular Enzymology, Graduate School of Bioengineering, Soka University, Tokyo, Japan.
authors.forename: Yota
authors.initials: Y
authors.lastname: Tatara
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshida
authors.forename: Eiji
authors.initials: E
authors.lastname: Ichishima
mesh_terms: D001230:Aspergillus / Q000201:enzymology*; D003545:Cysteine; D004220:Disulfides; D004795:Enzyme Stability; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation
chemical_list: D004220:Disulfides; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase; D003545:Cysteine