title: [Studies on the mechanism of thermostability and thermophilicity change of thermostable alkaline phosphatase and its mutants].
abstract: The relationship among the substituted amino acids, the 3D structure simulated on PC through CPHmodels Server ( http://www.cbs.dtu. dk/services/CPHmodels/) and the thermostable performance of 4 thermostable alkaline phosphatase(TAP) mutants selected from a clone bank of more than 200 mutants were analyzed to explore the mechanism of thermostability change. These mutants are TAP(A410T) (A410-->T), TAP(P396S) (P396-->S), TAP2(N100S T320-->I) and TAP4(N100-->S P396-->S A410 -->V P490-->S). TAP and the mutants' thermostable performance was evaluated by measuring the highest tolerable temperature (T1/2) and the optimal reaction temperature (Topt). The 3D structure neighboring the substituted amino acids was simulated by Swiss-PDBViewer to observe the relationship between the structure change and the thermostable performance of TAP and its mutants. The results displayed that all these amino acid substitutions except the T320-->I mutant brought about only a little local change on TAP's 3D structure and very little effect on their optimal reaction temperature, but a significant decrease (nearly 10 degrees C) on their highest tolerable temperature. However, the T320-->I mutation due to close to TAP's active sites did bring about a significant descendents of the mutant in both the highest tolerable temperature and the optimal reaction temperature. Thus, it seems to be able to conclude that most of the amino acid substitutions, no matter where they locate and what structure change they may make, can cause TAP's highest tolerable temperature reduced significantly. What's more, if the mutation occurring near or in the active sites, it can also cause TAP's optimal reaction temperature reduced significantly at the same time.
authors.affiliation: College of Life Sciences, Nanjing Normal University, Nanjing 210097, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Yu
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Xu
authors.forename: Zhe
authors.initials: Z
authors.lastname: Jin
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D000469:Alkaline Phosphatase