title: A comprehensive alanine-scanning mutagenesis study reveals roles for salt bridges in the structure and activity of Pseudomonas aeruginosa elastase.
abstract: The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (kcat/Km), half-lives (t1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Fei
authors: F
authors: Bian
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Shousong
authors: S
authors: Yue
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Zhenying
authors: Z
authors: Peng
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Xiaowei
authors: X
authors: Zhang
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Gao
authors: G
authors: Chen
authors: Shandong Crop Germplasm Center, Shandong Academy of Agricultural Sciences, Jinan, China.
authors: Jinhui
authors: J
authors: Yu
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan, China.
authors: Ning
authors: N
authors: Xuan
authors: Biotechnology Research Center, Shandong Academy of Agricultural Sciences, Jinan, China; Graduate Education Center, Shandong Academy of Agricultural Sciences, Jinan, China.
authors: Yuping
authors: Y
authors: Bi
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D004795:Enzyme Stability; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D012492:Salts / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D012492:Salts; D010196:Pancreatic Elastase; D000409:Alanine