Biochemical characterization of an inverting S/O-HexNAc-transferase and evidence of S-linked glycosylation in Actinobacteria.

Yogita Sharma, Shimona Ahlawat, Alka Rao

Glycobiology 2022 Mar 19

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Antimicrobial peptides harboring S- and or O-linked glycans are known as glycocins. Glycocins were first discovered and best characterized in Firmicutes. S-glycosylation is an enzymatic process catalyzed by S-glycosyltransferases of the GT2 family. Using a heterologous expression system, here we describe an inverting S/O-HexNAc-transferase (SvGT), encoded by ORF AQF52_3101 of Streptomyces venezuelae ATCC 15439, along with its acceptor substrate (SvC), encoded by ORF AQF52_3099. Using in vitro and in vivo assays, we define the distinct donor specificity, acceptor specificity, regioselectivity, chemoselectivity and Y(G/A/K/Q/E ≠ ΔG)(C/S/T ≠ Y/N)(G/A ≠ P/Q)G as the minimum acceptor sequon of SvGT. Although UDP-GlcNAc served as the donor in the cellular milieu, SvGT could also utilize UDP-Glc and UDP-GalNAc as donors in vitro. Using mass spectrometry and western blotting, we provide evidence that an anti-O-GlcNAc antibody (CTD110.6) cross-reacts with S-GlcNAc and may be used to detect S-GlcNAcylated glycoconjugates directly. With an understanding of enzyme specificities, we finally employed SvGT to generate two proof-of-concept neoglycocins against Listeria monocytogenes. In conclusion, this study provides the first experimental evidence for S-glycosylation in Actinobacteria and the application of its S/O-HexNAc-transferase in glycocin engineering. © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Citation

Yogita Sharma, Shimona Ahlawat, Alka Rao. Biochemical characterization of an inverting S/O-HexNAc-transferase and evidence of S-linked glycosylation in Actinobacteria. Glycobiology. 2022 Mar 19;32(2):148-161