biostudies-literatureUnknown4(1)Gerloff DLFonds National de la Recherche LuxembourgA 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.STAR protocols101905https://www.ebi.ac.uk/biostudies/studies/S-EPMC9792956biostudies-literaturePrediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering.PMC9792956Mata Salcedo UCialini CIlina EIMuller TGerloff DLMittelbronn MfalsePrediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering.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).¹.2023-01-01T00:00:00Z2023 Mar2025-04-05T14:54:59.81Z2024-11-13T09:37:14.349ZS-EPMC97929563652885610.1016/j.xpro.2022.101905