A front-face 'S_Ni synthase' engineered from a retaining 'double-S_N2' hydrolase

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Organic Chemistry

Background:

• S_Ni-like mechanisms, characterized by front-face leaving group departure and nucleophile approach, are known in chemical and enzymatic substitutions of α-glycosyl electrophiles.
• Substitution pathways like S_Ni-like, S_N1, and S_N2 can have comparable energetics, suggesting the possibility of engineered pathway switching.

Purpose of the Study:

• To engineer the Sulfolobus solfataricus β-glycosidase, which naturally performs double S_N2 substitution, to adopt an S_Ni-like mechanism.
• To develop a β-stereoselective catalyst for glycoside synthesis from activated substrates.

Main Methods:

• Site-directed mutagenesis (E387Y mutation) to disrupt the native nucleophile.
• Enzyme characterization through pH profiling, kinetic analysis, and mechanism-based inactivators.
• Structural analysis using X-ray crystallography.
• Computational studies including metadynamics simulations and QM/MM free-energy landscape calculations.

Main Results:

• Mutation E387Y transformed the enzyme's catalytic mode from S_N2 to S_Ni-like, enabling β-stereoselective glycoside synthesis without a native nucleophile.
• Substrate recruitment via π-sugar interaction was identified as a key feature.
• The QM/MM free-energy landscape revealed similarities to natural S_Ni-like glycosyltransferases.

Conclusions:

• Engineered S_Ni-like pathways are feasible in β-glycosyltransfer enzymes with appropriate environmental modifications.
• The study demonstrates the potential for engineering novel catalytic mechanisms in enzymes.
• The findings suggest that 'β-S_Ni' mechanisms could be relevant for natural glycosyltransfer enzymes.