Role of N-Terminal Extensional Long α-Helix in the Arylesterase from Lacticaseibacillus rhamnosus GG on Catalysis and Stability

Abstract

In the α/β hydrolases superfamily, the extra module modulated enzymatic activity, substrate specificity, and stability. The functional role of N-terminal extensional long α-helix (Ala2-Glu29, designated as NEL-helix) acting as the extra module in the arylesterase LggEst from Lacticaseibacillus rhamnosus GG had been systemically investigated by deletion mutagenesis, biochemical characterization, and biophysical methods. The deletion of the NEL-helix did not change the overall structure of this arylesterase. The deletion of the NEL-helix led to the shifting of optimal pH into the acidity and the loss of thermophilic activity. The deletion of the NEL-helix produced a 10.6-fold drop in catalytic activity towards the best substrate pNPC10. NEL-Helix was crucial for the thermostability, chemical resistance, and organic solvents tolerance. The deletion of the NEL-helix did not change the overall rigidity of enzyme structure and only reduced the local rigidity of the active site. Sodium deoxycholate might partially replenish the loss of activity caused by the deletion of the NEL-helix. Our research further enriched the functional role of the extra module on catalysis and stability in the α/β hydrolase fold superfamily.