| P-nitrophenylphosphatase (pNPPase) is an Mg2+ -dependent enzyme. The single ester bond of pNPP is cleaved by pNPPase in the reaction yielding phosphate and p-nitrophenol (pNP). Thermo p-nitrophenylphosphatase (TpNPPase) from Geobacillus stearothermophlius has the advantages of resistance to heat and organic solvents. Identifying molecular mechanisms involved in stability of thermophilic proteins is valuable to protein engineering in both scientific and industrial fields. Since a large amount of mutants with either increased or decreased thermostabilities can be obtained and analyzed systematically, the moderately themo-stable feature of TpNPPase makes it an ideal material to study the mechanism of protein thermostabilization.In this study, we report the X-ray structures of the H170Y and Q173G mutant proteins, which are obtained by directed evolution with improved thermostability. T1/2 and Tm values of H170Y and Q173G appear to be higher than that of wildtype. The T1/2 and Tm value have increased by 3.28℃ and 2.39℃ of H170Y, as well as 3.27℃ and 3.00℃ of Q173G, respectively.The proteins were crystallized by using the hanging-drop vapor-diffusion method and the data of X-ray diffraction were collected to the resolution of 2.0 A (H170Y) and 1.9 A (Q173G). The structures of H170Y and Q173G were obtained by molecular replacement, based on the structure of the wildtype. In the structure of H170Y, the presence of a sodium ion and a sulfate ion in the active site was found. The cap domain of chain B shows a half-closed conformation.An additional hydrophobic interaction was found through PIC webserver with the interaction distance of 4.6 A, and might be the key factor to the thermostabilization of H170Y. Besides, the reduction of the ASA might be the key factor to the thermostabilization of Q173G. This study potentially offered a molecular basis for both investigation into the catalytic mechanism and thermo-stable protein engineering. |
PDF Full Text Request