Year: 2026
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide 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 p K a 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 N π -methyl- l -histidine (πMH) resulted in substantial enzyme yields, efficient metal coordination for either Zn 2+ or Co 2+, and up to 5-fold improved tolerance to acidic conditions. Detailed mechanistic analysis revealed a systematic decrease in catalytic p K a 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.
Source: Biochemistry
Institution: University of Zurich
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W7130316259