Chemical Rescue Serves as a Predictive Proxy for Glycosynthase Activity on Glycosidic Bonds via a Shared Glycosyl Oxocarbenium Transition State

Abstract Engineered glycosynthases (GSs) are powerful biocatalysts for custom glycan synthesis, yet their optimization via directed evolution is severely constrained by bottlenecks in high-throughput screening for activated azido-sugar donors. Here, we demonstrate that chemical rescue (CR)—the azide-mediated restoration of hydrolytic activity in nucleophile-deficient mutants—serves as a predictive, high-throughput proxy for glycosynthase activity. Applying an azide-responsive Escherichia coli biosensor screen to a site-saturation mutagenesis library of Thermotoga maritima α -L-fucosidase ( TmAfc ), we established a strong rank-order correlation between CR and GS activities in both crude lysates ( ρ = 0.73) and purified enzymes ( ρ = 0.95). Transition path sampling and QM/MM umbrella sampling revealed that both pathways proceed through a shared oxocarbenium-ion-like transition state (Δ G ‡ ≈ 8.7 kcal/mol), providing a structural and thermodynamic rationale for using CR to select for transition-state-stabilizing mutations. Biochemical characterization of top-performing variants yielded an engineered fucosynthase (TmAfc_D224G_N70D_T392S) exhibiting a nearly 100-fold enhancement in V max alongside altered regioselectivity. This two-tiered screening framework leverages cost-effective chemical rescue assays to streamline glycosynthase engineering for tailored glycans synthesis.