TY - JOUR
T1 - New insights into the role of T3 loop in determining catalytic efficiency of GH28 endo-polygalacturonases
AU - Tu, Tao
AU - Meng, Kun
AU - Luo, Huiying
AU - Turunen, Ossi
AU - Zhang, Lujia
AU - Cheng, Yanli
AU - Su, Xiaoyun
AU - Ma, Rui
AU - Shi, Pengjun
AU - Wang, Yaru
AU - Yang, Peilong
AU - Yao, Bin
AU - Dobson, Renwick
N1 - Publisher Copyright:
© 2015 Tu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2015/9/1
Y1 - 2015/9/1
N2 - 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 muta-genesis 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 Δ(ΔG) 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.
AB - 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 muta-genesis 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 Δ(ΔG) 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.
UR - https://www.scopus.com/pages/publications/84943261147
U2 - 10.1371/journal.pone.0135413
DO - 10.1371/journal.pone.0135413
M3 - 文章
C2 - 26327390
AN - SCOPUS:84943261147
SN - 1932-6203
VL - 10
JO - PLoS ONE
JF - PLoS ONE
IS - 9
M1 - e0135413
ER -