A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries

Area of Science:

• Biochemistry
• Enzymology
• Protein Engineering

Background:

• Directed evolution and saturation mutagenesis are key for improving enzyme properties like substrate selectivity and stability.
• High-throughput assays are crucial for analyzing enzyme libraries, but suitable methods for glycosyltransferases are scarce.
• The blood group alpha-(1-->3)-galactosyltransferase (GTB) and alpha-(1-->3)-N-acetylgalactosaminyltransferase (GTA) are homologous enzymes involved in synthesizing blood group antigens.

Purpose of the Study:

• To develop a general, high-throughput screening assay for glycosyltransferases.
• To apply this assay for directed evolution of GTB to enhance its UDP-GalNAc transfer activity.
• To identify and characterize novel GTB mutants with improved enzymatic properties.

Main Methods:

• A novel screening assay was developed using GTB as a model, based on the pH indicator bromothymol blue.
• Saturation mutagenesis was performed on the GTB enzyme at residue M214, a hot spot near a metal-binding motif.
• The resulting enzyme library was screened for increased UDP-GalNAc transfer activity using the pH indicator assay.

Main Results:

• Two novel GTB mutants, M214G and M214S, were identified through pH indicator screening.
• Purified M214S and M214G mutants exhibited a two-fold increase in k(cat) and specific activity for UDP-GalNAc compared to wild-type GTB.
• The study confirmed the critical role of residue M214 in determining donor enzyme specificity.

Conclusions:

• A robust and general screening assay for glycosyltransferases has been successfully developed.
• The directed evolution approach using this assay identified GTB mutants with significantly enhanced activity towards UDP-GalNAc.
• The findings highlight the importance of residue M214 in GTB for substrate specificity and provide a foundation for engineering other glycosyltransferases.