title: Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases.
abstract: Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Tms increased by 0.6-3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Tao
authors.initials: T
authors.lastname: Tu
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Yeqing
authors.initials: Y
authors.lastname: Li
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Su
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Rui
authors.initials: R
authors.lastname: Ma
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
authors.affiliation: Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou 571100, P. R. China.
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002412:Cations; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis / Q000235:genetics*; D010368:Pectins / Q000737:chemistry / Q000378:metabolism; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology; D011096:Polygalacturonase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D000071436:Thermotolerance
chemical_list: D002412:Cations; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid