Tu Tao, Meng Kun, Luo Huiying, Turunen Ossi, Zhang Lujia, Cheng Yanli, Su Xiaoyun, Ma Rui, Shi Pengjun, Wang Yaru, Yang Peilong, Yao Bin
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, P. R. China.
Department of Biotechnology and Chemical Technology, School of Chemical Technology, Aalto University, FI-00076, Aalto, Finland.
PLoS One. 2015 Sep 1;10(9):e0135413. doi: 10.1371/journal.pone.0135413. eCollection 2015.
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.
柔性环的分子内流动性和构象变化在蛋白质的结构和功能完整性中起着重要作用。糖苷水解酶（GH）家族28的嗜热栖热放线菌Xz8内切聚半乳糖醛酸酶（PG8fn）以其高催化活性（28,000 U/mg）而著称。结构建模表明，PG8fn具有一个柔性的T3环，该环部分折叠在活性位点的底物上方，并通过活性位点裂隙中高度保守的残基Asn94与底物形成氢键。我们的研究调查了位于底物一侧催化残基上方的T3环中Asn94的催化作用。对突变体N94A进行的分子动力学模拟显示，由五聚半乳糖醛酸的O34处的羟基与Asn94至关重要的ND2形成的氢键丧失，以及底物随之从活性位点脱离和旋转，而N94Q突变体则由于侧链较长导致底物从其位置漂移开。与模拟结果一致，该位点的定点诱变表明该位置对氨基酸取代非常敏感。除了Km值从0.32（野生型PG8fn）改变为0.75 - 4.74 mg/ml外，所有突变体的kcat（约3 - 20,000倍）和kcat/Km（约8 - 187,500倍）值均显著降低，△(△G)值显著增加（5.92 - 33.47 kJ/mol）。综上所述，GH28 T3环中的Asn94在将底物以正确方式定位到靠近催化残基的位置方面起着关键作用。
