{
  "abstract": "Infant-associated bifidobacteria utilize lacto-N-biose I (LNB) and galacto-N-biose (GNB), major components of human milk oligosaccharides and intestinal mucin, respectively, through the GNB/LNB metabolic pathway. A UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) from Bifidobacterium longum JCM 1217 (BlGalT2) catalyzes a key step in this pathway. BlGalT2 belongs to class II GalTs within the histidine triad (HIT) superfamily, a rare protein group distinct from canonical class I GalTs. Here, we determined the crystal structure of BlGalT2 at 2.25 [A] resolution, providing the first three-dimensional structure of a class II GalTs. BlGalT2 is a monomeric enzyme with an architecture distinct from canonical class I GalT, comprising characteristic HIT1 and HIT2 catalytic core subdomains and an extended auxiliary domain that forms a self-contained active site. Kinetic analysis revealed a strong preference for GalNAc-1P over Gal-1P, with a 50-fold lower Km for GalNAc-1P, consistent with a hydrophobic pocket accommodating the N-acetyl group. Metal analysis and activity measurements indicated that Zn2+ is required for full activity, and structural prediction suggested its coordination by residues including the first histidine of the HIT motif. AlphaFold3 prediction and mutational analysis identified residues involved in nucleotide-sugar recognition and supported a Ping-Pong reaction mechanism involving a covalent enzyme-uridine monophosphate intermediate. These findings reveal how class II GalTs have evolved a unique structural framework and substrate specificity for N-acetylated sugars. Moreover, the structure of BlGalT2 fills the final missing gap in bifidobacterial HMO metabolism and provides molecular insight into the adaptation of bifidobacteria to host-derived glycans.",
  "abstract_shown": true,
  "affiliations": [
    "The University of Tokyo"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 5,
  "authors": [
    {
      "name": "M. Sato"
    },
    {
      "name": "C. Yamada"
    },
    {
      "name": "M. Nishimoto"
    },
    {
      "name": "M. Kitaoka"
    },
    {
      "affs": [
        0
      ],
      "name": "S. Fushinobu"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/ej5nwrrhe4e1a.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.13.751320",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1.full",
  "id": "ej5nwrrhe4e1a",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "biochemistry"
    ],
    [
      "openalex",
      "2505|Enzyme Structure and Function"
    ]
  ],
  "openalex": "W7213321676",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-13",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/ej5nwrrhe4e1a.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:22:39Z",
      "native_id": "10.64898/2026.09.13.751320",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1"
    }
  ],
  "subjects": [
    {
      "field": "chemistry",
      "origin": "openalex:2505|Enzyme Structure and Function",
      "subfield": "materials-polymers"
    }
  ],
  "title": "Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism",
  "title_text": "Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism",
  "type": "preprint",
  "updated_at": "2026-10-30T12:22:39Z",
  "url": "https://research.draconitic.com/paper/ej5nwrrhe4e1a",
  "venue": "bioRxiv",
  "versions": []
}