title: Structure of a His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus.
abstract: Using directed evolution based on random mutagenesis and heat-treated selection, a thermostable His170Tyr mutant of Geobacillus stearothermophilus thermostable p-nitrophenylphosphatase (TpNPPase) was obtained. The temperature at which the His170Tyr mutant lost 50% of its activity (T1/2) was found to be 4.40 K higher than that of wild-type TpNPPase, and the melting temperature of the His170Tyr mutant increased by 2.39 K. The crystal structure of the His170Tyr mutant was then determined at 2.0 Å resolution in the presence of a sodium ion and a sulfate ion in the active site. The cap domain of chain B shows a half-closed conformation. The hydrophobic side chain of the mutated residue, the hydroxyphenyl group, forms a hydrophobic contact with the methyl group of Ala166. This hydrophobic interaction was found using the Protein Interactions Calculator (PIC) web server with an interaction distance of 4.6 Å, and might be a key factor in the thermostabilization of the His170Tyr mutant. This study potentially offers a molecular basis for both investigation of the catalytic mechanism and thermostable protein engineering.
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China.
authors.forename: Tiantian
authors.initials: T
authors.lastname: Shen
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, People's Republic of China.
authors.forename: Chaoneng
authors.initials: C
authors.lastname: Ji
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry*; D009154:Mutation; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D014443:Tyrosine; D006639:Histidine; D009597:4-Nitrophenylphosphatase
keywords: TpNPPase; directed evolution; random mutagenesis; thermostability