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

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.

GLYCOCINS: The sugar peppered antimicrobials.

Glycosylated bacteriocins, known as glycocins, were first discovered in 2011. These bioactive peptides are produced by bacteria to gain survival advantages. They exhibit diverse types of glycans and demonstrate varied antimicrobial activity. Currently, there are 13 experimentally known glycocins, with over 250 identified in silico across different bacterial phyla. Notably, glycocins are recognized for their glycan-mediated antimicrobial activity, proving effective against drug-resistant and foodborne pathogens. Many glycocins contain rare S-linked glycans. Glycosyltransferases (GTs), responsible for transferring sugar to glycocins and involved in glycocin biosynthesis, often cluster together in the producer's genome. This clustering makes them valuable for custom glycoengineering with diverse substrate specificities. Heterologous expression of glycocins has paved the way for the establishment of microbial factories for glycopeptide and glycoconjugate production across various industries. In this review, we emphasize the primary roles of fully and partially characterized glycocins and their glycosylating enzymes. Additionally, we explore how specific glycan structures facilitate these functions in antibacterial activities. Furthermore, we discuss newer approaches and increasing efforts aimed at exploiting bacterial glycobiology for the development of food preservatives and as replacements or complements to traditional antibiotics, particularly in the face of antibiotic-resistant pathogenic bacteria.

Global shape of SvGT, a metal-dependent bacteriocin modifying S/O-HexNActransferase from actinobacteria: c -terminal dimerization modulates the function of this GT.

Here, we describe hitherto unknown shape-function of S/O-HexNActransferase SvGT (ORF AQF52_3101) instrumental in glycosylation of bacteriocin SvC (ORF AQF52_3099) in Streptomyces venezuelae ATCC 15439. Data from gel filtration, mass spectrometry, analytical ultracentrifugation, and Small Angle X-ray Scattering (SAXS), experiments confirmed elongated dimeric shape in solution for SvGT protein. Enzyme assays confirmed the dependence of SvGT on the availability of Mg2+ ions to be functionally activated. SAXS data analysis provided that apo and Mg2+-activated protein adopt a shape characterized by a radius of gyration and maximum linear dimension of 5.2 and 17.0 nm, and 5.3 and 17.8 nm, respectively. Alphafold2 server was used to model the monomeric chain of this protein which was docked on self to obtain different poses of the dimeric entity. Experimental SAXS data was used to select and refine the structure of SvGT dimer. Results showed that Mg2+ ions induce reorientation of the GT domain of one chain leading to a dimer with C2 symmetry, and the C-terminal portion entangles with each other in all states. Mutation-rendered alteration in activity profiles confirmed the role of conserved residues around catalytic motif. Global structure analysis puts forth the need to understand the role of constitutionally diverse C-terminal portion in regulating substrate selectivity.Communicated by Ramaswamy H. Sarma.