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. 2024 Jun 11;12(6):e2472. doi: 10.1002/mgg3.2472

FIGURE 6.

FIGURE 6

Activity of recombinant Ile186Asn‐UXS1 and WT‐UXS1. Purified recombinant enzymes were incubated with UDP‐GlcA in the presence of NAD+. Analysis of nucleotide sugars was carried out by PGC‐LC‐ESI‐MS/MS. (a) Extracted ion chromatogram at m/z 535 and 579, corresponding to deprotonated UDP‐Xyl and UDP‐GlcA, respectively. Only WT‐UXS1 converts UDP‐GlcA to UDP‐Xyl. (b) MS/MS spectrum of precursor ions at m/z 579.09 (upper) and 535.00 (lower). Fragmentation ions at m/z 211, 323, and 403 are consistent with predicted fragmentation of a UDP‐sugar into [UDP‐H−]−, [UMP‐H−]−, and [UDP‐uridine‐H−]−, respectively. (c) Ion intensity of ions at m/z 535 and 579. The decrease of UDP‐GlcA is commensurate with increased UDP‐Xyl reflecting the activity of WT enzyme. With mutant enzyme, only a decrease of UDP‐GlcA was observed.