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 (ΔG‡ ≈ 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 Vmax alongside altered regioselectivity. This two-tiered screening framework leverages cost-effective chemical rescue assays to streamline glycosynthase engineering for tailored glycans synthesis.
Mohit Kumar, C. Bandi, Sri Vidya Vyjayanthi Tallavajhula et al.· bioRxiv· 0 citations
Aberrant activation of Wnt signaling results in unregulated accumulation of cytosolic β-catenin, which subsequently enters the nucleus and promotes transcription of genes that contribute to cellular proliferation and malignancy. Here, we sought to eliminate pathogenic β-catenin from the cytosol using designer ubiquibodies (uAbs), chimeric proteins composed of an E3 ubiquitin ligase and a target-binding domain that redirect intracellular proteins to the proteasome for degradation. To accelerate uAb development, we leveraged a protein language model–driven algorithm called SaLT&PepPr to computationally design “guide” peptides with affinity for β-catenin, which were subsequently fused to the catalytic domain of a human E3 called carboxyl terminus of Hsp70-interacting protein. Expression of the resulting peptide-guided uAbs in colorectal cancer cells led to the identification of several designs that greatly reduced the abnormally stable pool of free β-catenin in the cytosol and nucleus while preserving the normal membrane–associated subpopulation. This selective knockdown of pathogenic β-catenin suppressed Wnt/β-catenin signaling and impaired tumor cell survival and proliferation. Furthermore, one of the best degraders selectively decreased cytosolic but not membrane-associated β-catenin levels in livers of BALB/c mice following delivery as a lipid nanoparticle–encapsulated mRNA. Collectively, these findings reveal the unique ability of uAbs to selectively deplete abnormal proteins in vitro and in vivo and open the door to peptide-programmable biologic modulators of other disease-causing proteins.
Tianzheng Ye, A. Alamgir, C. Robertus et al.· Science Advances· 0 citations
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