Catalytic Residue Reprogramming Enhances Enzyme Activity at Alkaline pH via Phenolate-Mediated Proton Transfer.

ABSTRACT: Achieving efficient enzyme catalysis under extreme pH conditions remains a major challenge in biocatalysis and synthetic biology. To address this, we present an enzyme engineering strategy that integrates rational redesign of catalytic residues with directed evolution to enable robust enzyme function at alkaline pH. The core principle involves replacing the conserved general base with an ionizable residue of higher intrinsic pK_a, shifting the proton transfer mechanism from carboxylate- to phenolate-mediated catalysis. Previously, we engineered TEM β-lactamase by substituting the universally conserved Glu166 with tyrosine (E166Y), which severely impaired activity. Directed evolution subsequently restored function, yielding the optimized variant YR5-2. Although this enginee

SUBMITTER: Vajanapanich P 

PROVIDER: S-EPMC12455642 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature