New Insights into the Role of T3 Loop in Determining Catalytic Efficiency of GH28 Endo-Polygalacturonases.
Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75-4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3-20,000 fold) and kcat/Km (~8-187,500 fold) values and significantly increased △(△G) values (5.92-33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Tao
T
Tu
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Kun
K
Meng
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Huiying
H
Luo
Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, FI-00076, Aalto, Finland.
Ossi
O
Turunen
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China.
Lujia
L
Zhang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Yanli
Y
Cheng
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Xiaoyun
X
Su
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Rui
R
Ma
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Pengjun
P
Shi
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Yaru
Y
Wang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Peilong
P
Yang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Bin
B
Yao
D001665:Binding Sites; D007700:Kinetics; D062105:Molecular Docking Simulation; D011096:Polygalacturonase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D020037:Sordariales / Q000201:enzymology; D013379:Substrate Specificity
D011096:Polygalacturonase