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.
Highlights
IntroductionThree-dimensional structures of ten endo-PGs are available (http://www.rcsb.org)
Because Lys and Tyr lead to favorable cation-πinteractions in thermophilic proteins[24], residues in close proximity to the 38 targets of modeled PG63 were substituted to Lys or Tyr in silico
The brute-force approach combined with 10-ns molecular dynamics (MD) simulations was used to examine the occupancy rate during the trajectory of each potential cation-πinteraction
Summary
Three-dimensional structures of ten endo-PGs are available (http://www.rcsb.org). These endo-PGs demonstrate similar three-dimensional structures, they vary in specificities, catalytic activity, pH preference, and thermostability. The catalytic process of enzymes includes 1) ligand binding to the active site of the enzyme; 2) hydrolysis of the glycoside bond; and 3) product release[18]. During this process, the enzyme molecule experiences different conformations of the enzyme-substrate complex and rapid transition to support maximum activity[19]. We first elucidated the important roles of cation-πinteraction in the catalytic process of endo-PG. This study deepens the understanding of the importance of this non-covalent interaction in enzyme catalysis and provides a novel strategy to improve GHs for greater catalytic performance
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