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.

SUBMITTER: Shen T 

PROVIDER: S-EPMC4051519 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature