Identification of active site residues in the Shigella flexneri glucosyltransferase GtrV

Abstract

The serotype-specific glucosyltransferase, GtrV, is responsible for glucosylation of the O-antigen repeating unit of Shigella flexneri serotype 5a strains. GtrV is an integral inner membrane protein with two essential periplasmic loops: the large Loop 2 and the C-terminal Loop 10. In this study, the full length of the Loop 2 was shown to be necessary for GtrV function. Site-directed mutagenesis within this loop revealed that conserved aromatic and charged amino acids have a critical role in the formation of the active site. Sequential deletions of the C-terminal end indicated that this region may be essential for assembly of the protein in the cytoplasmic membrane. The highly conserved FWAED motif is thought to form the substrate-binding site and was found to be critical in GtrV and GtrX, a serotype-specific glucosyltransferase with homology to GtrV. The data presented constitutes a targeted analysis of the formation of the GtrV active site and highlights the essential role of the large periplasmic Loop 2 in its function.

Keywords::

• Active site
• glucosyltransferase
• Shigella
• integral protein

Acknowledgements

JAM thanks F. Tavares for constant support during the Masters program.

Declaration of interest: This work was supported by a grant from the National Health and Medical Research Council of Australia (Grant number 366743). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.