{
      "number": 1,
      "subjects": [
        "QR"
      ],
      "eprintid": 3350,
      "date": 2024,
      "userid": 2,
      "rev_number": 9,
      "creators": [
        {
          "name": {
            "lineage": null,
            "given": "Sonali",
            "honourific": null,
            "family": "Sunsunwal"
          }
        },
        {
          "name": {
            "lineage": null,
            "given": "Aasawari",
            "honourific": null,
            "family": "Khairnar"
          }
        },
        {
          "name": {
            "lineage": null,
            "given": "Srikrishna",
            "honourific": null,
            "family": "Subramanian"
          }
        },
        {
          "name": {
            "lineage": null,
            "given": "T N C",
            "honourific": null,
            "family": "Ramya"
          }
        }
      ],
      "dir": "disk0\/00\/00\/33\/50",
      "keywords": "GlfM; Maf; O-glycosylation; bipartite; evolution; flagellin; flagellum; pseudaminic acid; specificity; tripartite.",
      "lastmod": "2026-02-07 12:09:57",
      "ispublished": "pub",
      "pagerange": 334,
      "publisher": "Cell Press",
      "metadata_visibility": "show",
      "date_type": "published",
      "eprint_status": "archive",
      "status_changed": "2026-02-07 12:09:57",
      "volume": 8,
      "datestamp": "2026-02-07 12:05:12",
      "uri": "http:\/\/crdd.osdd.net\/open\/id\/eprint\/3350",
      "refereed": "TRUE",
      "publication": "Commun. Biol.",
      "type": "article",
      "title": "Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras",
      "abstract": "Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine\/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence\/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5’-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser\/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO)."
    }