N-glycosylation-modifications-driven conformational dynamics attenuate substrate inhibition of d-lactonohydrolase.

Achieving enzyme catalysis at high substrate concentrations is a substantial challenge in industrial biocatalysis, and the role of glycosylation in post-translational modifications that modulate enzyme substrate inhibition remains poorly understood. This study provides insights into the role of N-glycosylation in substrate inhibition by comparing the catalytic properties of d-lactonohydrolase (d-Lac) derived from Fusarium moniliforme expressed in prokaryotic and eukaryotic hosts. Experimental evidence indicates that recombinant d-Lac expressed in Pichia pastoris (PpLac-WT) exhibits higher hydrolysis rates at a substrate concentration of 400 g/L, with reduced substrate inhibition and enhanced stability compared to the recombinant d-Lac expressed in Escherichia coli (EcLac-WT). Mutant PpLac-M1 achieves a conversion rate of 40 % at a substrate concentration of 400 g/L, with a space-time yield of d-pantoic acid reaching 91.1 g/L/h. Proteomics analysis reveals that residues N29 and N278, located approximately 10-20 Å from the active site undergo N-glycosylation in PpLac-WT. Using microsecond-scale molecular dynamics simulations and Markov state models, we elucidate the effects of glycosylation on the conformational flexibility of two key loops at the entrance of the binding pocket. Specifically, the loops in PpLac-WT can transition between open and closed states, whereas those in EcLac-WT tend to remain open. In high substrate concentration conditions, the open state causes congestion, leading to substrate inhibition. Shortest-path map analysis confirms that substrate entry is dynamically controlled by residue N29 on the loops surrounding the active site. Our findings enhance the understanding of the effects of glycosylation on enzyme conformational dynamics and provide insights into mitigating inhibition at high substrate concentrations.