Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS‐5

Abstract

EG5C‐1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate‐binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary‐coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative‐saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild‐type (WT) to 5678 U/µmol towards carboxymethyl cellulose‐Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (Km) and a remarkable improvement in catalytic efficiency (kcat/Km). Furthermore, molecular dynamics simulations revealed that the K51T/L93T mutation altered the spatial conformation at the active site cleft, enhancing the interaction frequency between active site residues and substrate, and improving catalytic efficiency and substrate affinity. The current studies provided some perspectives on the effects of distal residue substitution, which might assist in the engineering of processive endoglucanase or other glycoside hydrolases.

Modulation of a Loop Region in the Substrate Binding Pocket Affects the Degree of Polymerization of Bacillus subtilis Chitosanase Products

“…Notably, mutants TJN and TJA exhibited pronounced fluctuation within region 195–210, where the extra sequence is added. This indicated that the loop alteration increases the flexibility of the enzyme, potentially hindering the entry of the substrate during the catalytic process . These observations align with alterations in the catalytic activity.…”

“…This indicated that the loop alteration increases the flexibility of the enzyme, potentially hindering the entry of the substrate during the catalytic process. 46 These observations align with alterations in the catalytic activity.…”

Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

“…This prospect has therefore engendered significant attention. 12,13 Compared to CBHs, much less is known about the structure and function of processive EGs. They belong almost exclusively to the glycoside hydrolase family 9 (GH9) of enzymes associated with the bacterial cellulolytic system.…”

Enhancing the Catalytic Activity of Type II L-Asparaginase from Bacillus licheniformis through Semi-Rational Design

“…HotSpot Wizard server can be used for mutation site identification and library design [ 17 ], which has predicted hotspots of mutations for increasing enzyme activity [ 18 , 19 , 20 ]. EVcoupling web server can provide evolutionary constraints (EC) to infer correlations between amino acids at different sequence positions [ 21 ] and affect enzyme structure and function [ 22 ], which has been proved to be an effective approach to improving catalytic activity [ 23 , 24 ]. However, enzymatic improvement of type II ASNase by rational design combing structural information and bioinformatics tools has been rarely reported.…”