Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

STAR Protoc. 2023 Mar 17;4(1):101905. doi: 10.1016/j.xpro.2022.101905. Epub 2022 Dec 17.


A significant number of proteins are annotated as functionally uncharacterized proteins. Within this protocol, we describe how to use protein family multiple sequence alignments and structural bioinformatics resources to design loss-of-function mutations of previously uncharacterized proteins within the glycosyltransferase family. We detail approaches to determine target protein active sites using three-dimensional modeling. We generate active site mutants and quantify any changes in enzymatic function by a glycosyltransferase assay. With modifications, this protocol could be applied to other metal-dependent enzymes. For complete details on the use and execution of this protocol, please refer to Ilina et al. (2022).1.

Keywords: Bioinformatics; Molecular Biology; Protein Biochemistry; Protein expression and purification; Sequence analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay*
  • Computational Biology
  • Glycosyltransferases / genetics
  • Mutation
  • Protein Engineering*


  • Glycosyltransferases