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<inf>a</inf>, 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 engineering effort originally aimed to validate a novel selection platform, the evolutionary trajectory of YR5-2 exemplifies our proposed strategy in the present study. Here, we characterize YR5-2 and its parental variants across a wide pH range. Steady-state kinetic analyses reveal a &gt; 3-unit shift in the optimal pH for k<inf>cat</inf>, with YR5-2 reaching 870 s^–1at pH 10.0, a k<inf>cat</inf>value comparable to that of the wild type at its optimal pH. Kinetic analyses of Y166E revertants, together with molecular dynamics simulations, support a mechanistic transition in which Tyr166 functions as the catalytic general base. In vivo experiments further demonstrate the utility of YR5-2 as a selectable marker by enabling recombinant protein expression in E. coli under alkaline growth conditions. This work establishes a broadly applicable framework for reprogramming enzyme catalytic mechanisms, particularly in hydrolases, to expand their operational pH range and unlock new opportunities in industrial and environmental biocatalysis.