A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system

Griffiths, Ryan, Pfalzgraf, Hans, Latousakis, Dimitris, Ashworth, Gareth, Dong, Changjiang, Hemmings, Andrew and Juge, Nathalie (2025) A structural basis for the strain-dependent UDP-sugar specificity of glycosyltransferase C from the Limosilactobacillus reuteri accessory secretion system. Acta crystallographica. Section D, Structural biology, 81. pp. 708-717. ISSN 2059-7983

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Abstract

The accessory secretion (aSec) system is a protein export pathway that is uniquely present in Gram-positive bacteria and is dedicated to the secretion of large, glycosylated cell wall-anchored adhesins called serine-rich repeat proteins (SRRPs). Strain-specific glycosylation of SRRPs has previously been reported in Limosilactobacillus reuteri and attributed to GtfC, a glycosyltransferase belonging to family 113, with LrGtfC100-23 from L. reuteri rat strain 100-23C showing specificity for UDP-Glc, while LrGtfC53608 from L. reuteri pig strain ATCC 53608, which differs at only ten amino-acid positions, shows a preference for UDP-GlcNAc. However, the structural basis underpinning GtfC sugar-donor specificity remains unclear. Here, we report X-ray crystal structures of the tetrameric LrGtfC100-23 in the apo form and its complexes with UDP and with the noncognate sugar donor UDP-N-acetylglucosamine (UDP-GlcNAc). Analysis of the LrGtfC100-23 structures identified candidate residues implicated in donor-sugar substrate specificity, which were supported by site-directed mutagenesis. Reciprocal swaps of candidate residues combined with thermal shift assays revealed that the W240C variant of LrGtfC100-23 could bind both UDP-sugar donors, while the P243S variant of LrGtfC53608 became specific for UDP-Glc, opening the door for glycoengineering approaches in bacteria.

Item Type:	Article
Additional Information:	Data availability Crystallographic data for the structures of LrGtfC100–23 reported in this article have been deposited in the RCSB Protein Data Bank and are available free of charge via the DOIs https://doi.org/10.2210/pdb9HTX/pdb, https://doi.org/ 10.2210/pdb9HUA/pdb and https://doi.org/10.2210/pdb9HU9/pdb for the apo form, the complex with UDP and the complex with UDP-GlcNAc, respectively.
Uncontrolled Keywords:	accessory secretion systems,crystal structure,glycosyltransferases,gtfc,structural biology ,/dk/atira/pure/subjectarea/asjc/1300/1315
Faculty \ School:	Faculty of Science > School of Biological Sciences
Related URLs:	http://www.scopus.com/inward/record.url?...
Depositing User:	LivePure Connector
Date Deposited:	16 Dec 2025 14:30
Last Modified:	19 Dec 2025 01:46
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101421
DOI:	10.1107/S2059798325008782

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