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Structure of a His170Tyr mutant of thermostable pNPPase from Geobacillus stearothermophilus
Author(s) -
Shen Tiantian,
Guo Zheng,
Ji Chaoneng
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14007341
Subject(s) - geobacillus stearothermophilus , mutant , thermostability , chemistry , mutagenesis , directed evolution , protein engineering , hydrophobic effect , stereochemistry , site directed mutagenesis , biochemistry , thermophile , enzyme , gene
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 ( T 1/2 ) 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.