Hydrolytic metalloenzymes employ Lewis-acidic metal cofactors to activate water molecules, generating nucleophilic hydroxide species that facilitate catalysis. Their catalytic efficiency across a wide pH range is often governed by the protonation state of the metal-bound water, reflected in pKa values typically between 6.8 and 9. Modulating this parameter is key to expanding enzymatic activity for improved activity at neutral to acidic pH. Herein, we apply genetic code expansion to mutate the primary metal-coordination sphere of a model metallohydrolase: the dizinc phosphotriesterase from Pseudomonas diminuta. Substitution of the most catalytically indispensable coordinating histidine residue (H55) to Nπ-methyl-l-histidine (πMH) resulted in substantial enzyme yields, efficient metal coordination for either Zn2+ or Co2+, and up to 5-fold improved tolerance to acidic conditions. Detailed mechanistic analysis revealed a systematic decrease in catalytic pKa and attenuation of several catalytic rate constants. These results add to the growing body of evidence demonstrating the power of ncAA-based engineering for refined tuning of enzyme properties.
Manser et al. (2026) studied this question.