Thermostability Engineering of a Chitin-Utilizing GlcNAc Deacetylase from Acinetobacter baumannii via Distal Mutations.
TL;DR
The deacetylase repertoire is expanded and provides a framework for engineering stable industrial enzymes and molecular dynamics simulations revealed that T93P reduces backbone flexibility, C153T enhances β-sheet rigidity via hydrogen bonding, and A244M improves hydrophobic packing by filling a core cavity.
Abstract
The deacetylation of N-acetylglucosamine (GlcNAc) to glucosamine (GlcN) is a key step in enzymatic chitin valorization. We identified a novel GlcNAc deacetylase (AbNGD) from Acinetobacter baumannii with high substrate specificity and catalytic activity but poor thermal stability (half-life of 5.5 h at 37 °C). Using the FireProt platform, we generated a combinatorial mutant, AbNGD-M3 (T93P-C153T-A244M). Its optimal temperature increased to 37 °C, and its half-life reached 28.1 h (5.1-fold improvement), while retaining 97% of wild-type catalytic efficiency (kcat/Km = 3.4 mM-1·s-1). Site-directed mutagenesis confirmed Asp54 and His160 as key catalytic residues. Molecular dynamics simulations revealed that T93P reduces backbone flexibility, C153T enhances β-sheet rigidity via hydrogen bonding, and A244M improves hydrophobic packing by filling a core cavity. These distal mutations collectively balance stability and activity. Substrate channel analysis identified a likely transport pathway centered on T1. This study expands the deacetylase repertoire and provides a framework for engineering stable industrial enzymes.