Fig. 5: Identification of catalytic residues in the N- and C-terminal GT domains of CS synthases.
From: Structural basis for human chondroitin sulfate chain polymerization
a, b Structural comparisons of the putative catalytic sites of the N-terminal GT31 domains and (b) the C-terminal GT7 domains of CHSY3 (colored in orange), CHSY1 (colored in green), CHPF (colored in gray), and CHPF2 (colored in blue). CHSY3 and CHPF structures were determined using cryo-EM, while CHSY1 and CHPF2 were predicted using AlphaFold 233,34. a A UDP molecule, representing a substrate analog or reaction product, bound to CHSY3, is shown in stick representation and colored in cyan, and the Mn2+ ion is shown as a purple sphere. Residues in CHSY3 important for UDP binding are highlighted in stick representation. b In the C-terminal GT domain, a structural superimposition with the chondroitin polymerase from Escherichia coli strain K4 in complex with UDP-GalNAc (colored in light blue) (PDB-ID: 2Z87)41 allowed us to propose the interactions with the donor substrate. All the residues identified to interact with the substrate donors are represented in sticks. c–f In vitro glycosylation assays for the CHSY3-CHPF complexes, followed by FACE analysis. Images shown are representative of three independent experiments. Source data is provided. c Activity of wild-type and CHSY3-mutant complexes, in which mutations are localized in the N-terminal GT domain. The pentasaccharide peptide Penta-CSF1 was used as the acceptor substrate, while both UDP-GalNAc and UDP-GlcA were used as donor substrates. d Activity of wild-type and CHSY3-mutant complexes with mutations in the C-terminal GT domain. The hexasaccharide peptide Hexa-CSF1 was used as the acceptor and UDP-GalNAc and UDP-GlcA were used as substrate donors. e Effect of different metal ion species on the GlcA and f GalNAc transferase reaction. For all the FACE gels, penta-, hexa-, and heptasaccharide peptides were loaded alongside as weight markers. Monosaccharide symbols follow the SNFG (Symbol Nomenclature for Glycans) system67. Source data are provided as a Source Data file.
