Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism
- 1The University of Tokyo
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.
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BibTeX
@misc{sato2026crystal,
title = {{Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism}},
author = {M. Sato and C. Yamada and M. Nishimoto and M. Kitaoka and S. Fushinobu},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.13.751320},
url = {https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Crystal structure of a class II UDP-glucose-hexose-1-phosphate uridylyltransferase from Bifidobacterium longum involved in human milk oligosaccharide metabolism
AU - M. Sato
AU - C. Yamada
AU - M. Nishimoto
AU - M. Kitaoka
AU - S. Fushinobu
PY - 2026
DA - 2026/09/13
PB - bioRxiv
DO - 10.64898/2026.09.13.751320
UR - https://www.biorxiv.org/content/10.64898/2026.09.13.751320v1
AB - 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.
ER -