Fig. 2: Functionally inactivated UXS1 requires reactivation by a UDP-4-keto sugar when H6PD is highly active.
From: A missing enzyme-rescue metabolite as cause of a rare skeletal dysplasia
a, Working hypothesis: an abortive catalytic cycle of UXS1 leads to the inactivation of the enzyme, which is counteracted by UDP-4-keto sugars. The decarboxylation of UDP-glucuronate by UXS1 depends on the oxidation of the C4 hydroxyl group using a tightly bound NAD+. This generates NADH and a UDP-4-ketoxylose intermediate. Normally, formation of UDP-xylose regenerates NAD+, preparing the enzyme for another cycle. Infrequently, the intermediate dissociates from the catalytic pocket, leaving UXS1 bound to NADH and inactive. We hypothesized that UDP-4-keto sugars restore activity by facilitating the oxidation of enzyme-bound NADH to NAD+. Loss of the cofactor can also yield an inactive apoenzyme, but reactivation by NAD+ binding is limited by the low NAD+/NADH ratio in the endoplasmic reticulum maintained by H6PD. b, Schematic of the experiment to test whether UDP-4-ketoxylose produced by ArnA can reactivate UXS1. c–f, ArnA expression rescues UDP-xylose synthesis in TGDS-KO cells, but not in UXS1-KO cells. UDP-xylose (c,e), UDP-glucuronate (d,f) were quantified by LC–MS in parental 293T (c,d) and HAP1 (e,f) cells, and in TGDS-KO or UXS1-KO clones transduced with a lentivirus expressing ArnA or an empty vector. ND, not determined. g, Experiment exploring the role of H6PD in UXS1 dependency on TGDS. h–k, H6PD modulates UXS1 dependency on TGDS. In HAP1 cells, CRISPR–Cas9-mediated H6PD knockdown (using two different guide RNAs (gRNAs) versus control gRNA (−)) reduces the effect of TGDS deficiency on UXS1 function (h,i). In U2OS cells, H6PD overexpression (+) makes UXS1 activity dependent on TGDS (j,k). −, empty vector control. UDP-xylose (h,j) and UDP-glucuronate (i,k) were quantified by LC–MS. Data are mean ± s.d. of 3 independent experiments, each containing 3 biological replicates. Paired two-tailed Dunnett (c,d) or Sidak (e,f,i–k) post hoc testing of log-transformed data after one-way ANOVA. For exact P values see Source Data.
