Structural basis and functional mining of active site loop for fine tuning substrate affinity of L-asparaginase from Bacillus licheniformis.

The widespread presence of the environmental contaminant acrylamide (AA) in thermally processed foods has been linked to serious environmental and health challenges. Although the formation of AA can be fundamentally inhibited by L-asparaginase (L-ASNase), its removal efficiency is highly dependent on substrate affinity. However, the structural mechanism by which the affinity of this L-ASNase is regulated remains poorly understood. Herein, a rational design strategy of active site loop (ASL) engineering was initially employed to improve the substrate affinity of L-ASNase from Bacillus licheniformis. As a result, two single mutants (K18S and L24Y) both exhibited approximately 50% increased substrate affinities with unaffected catalytic activities. Moreover, the combined double mutant K18S/L24Y achieved a significantly lower Km value of 1.49 ± 0.25 mM. Subsequently, structural analysis revealed that mutants K18S and L24Y obtained disordered active site loops (ASLs) compared with the β-hairpin structure in wild type by reducing the hydrogen bonds and changing the hydrophobic interactions, displaying the altered interaction forces between amino acids on the active site for substrate binding. Building on these structural observations, molecular dynamics simulations were further performed to uncover the underlying physical mechanism. It was revealed by the simulations that the mutations lead to reduced dynamic fluctuations and the achievement of an energetically neutral state in the ASL region, thereby converting substrate binding from a "high-resistance" mode to a "zero-resistance" mode and consequently enhancing affinity. Additionally, mutant K18S held the same α-helix orientation linked to ASL as the wild type for maintaining catalytic activity. To our knowledge, this study fills a gap in the structural mechanism of substrate affinity for L-ASNases and offers a guideline for engineering L-ASNases from different origins for environmental contaminant applications.