Extended Data Fig. 5: Products from the bacterial and fungal orthologue of TGDS reactivate inactivated UXS1.
From: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia
a, Extracted ion chromatograms for the m/z corresponding to UDP-xylose, UDP-4-keto-6-deoxyglucose (‘UDP-4k6dG’), UDP-6-deoxyhexose (‘UDP-6dH’), NAD+, and NADH, obtained by LC-MS in reactions where UDP-glucuronate was incubated with UXS1, with sodium-borohydride-inactivated UXS1, or with inactivated UXS1 in the presence of the product of human TGDS (‘TGDS-P’). Quantifications as in Fig. 3h, but including a condition where 0.4 µM NAD+ was added, demonstrating that low levels of free NAD+ present in the TGDS preparation do not suffice to rescue UDP-xylose production. b-c, Experiment described in panel a but using the Botrytis cinerea UG46DH product (‘UG46DH-P’, b) or E. coli ArnA product (‘ArnA-P’, c). d, Schematic representation of the formation of different NADH forms as well as the quantification of 1,6-NADH and 1,2-NADH in the experiment presented in Fig. 3h. e, Quantification of UDP-xylose production in an experiment as described in Fig. 3h, but including a condition where 10 µM NAD+ was added, demonstrating that UXS1 activity can be restored by sufficient amounts of NAD+. Data are means of two (e) or means ± SD of three independent experiments, each consisting of three (a,d) or one biological replicate (b,c). * p < 0.05, ** p < 0.01, *** p < 0.001 in two-tailed paired Dunnett post-hoc testing of log-transformed data after one-way ANOVA. For exact p-values see source data file.
