Structural instability impairs function of the <scp>UDP</scp> ‐xylose synthase 1 <scp>Ile181Asn</scp> variant associated with short‐stature genetic syndrome in humans

Glycosaminoglycan assembly on proteoglycans involves a common tetrasaccharide linker that starts with xylose attached to a serine on the protein. Defective linker biosynthesis caused by a missense mutation of human UDP-xylose synthase (hUXS1) is associated with connective tissue disorders characterized by skeletal abnormality and short stature. The Ile181Asn variant of hUXS1 was reported as inactive in releasing UDP-xylose from UDP-glucuronic acid. Here, we show that Ile181Asn-hUXS1 exhibited catalytic properties similar to the wild-type enzyme but featured a significant decrease in stability, expressed in melting temperature lowered from 48.2 °C to 35.2 °C. At 37 °C, Ile181Asn-hUXS1 was ~10-fold less stable and more prone to precipitation than wild-type hUXS1. The loss of function in Ile181Asn-hUXS1 is thus explained by instability, consistent with molecular dynamics simulations predicting structural destabilization. Impact statement The Ile181Asn variant of human UDP-xylose synthase (hUXS1), associated with a short-stature genetic syndrome, has previously been reported as inactive. We show here with experiments and molecular simulations that hUXS1 malfunction arises from structural instability rather than from a catalytic defect.