UDP-glucose dehydrogenase (UDGH) catalyzes the NAD+ dependent oxidation of UDP-glucose to UDP-glucuronic acid. In humans, (h)UGDH is regulated by the feedback inhibitor UDP-xylose by an atypical allosteric mechanism. Briefly, UDP-xylose competes with UDP-glucose for the active site. Binding UDP-xylose triggers a conformational change from an active 32 symmetry hexamer (E) to an inhibited horseshoe shaped complex (EΩ). The ligand induced conformational change is the result of altering the affinity of the interface between subunits, i.e. allostery. hUGDH also displays hysteresis, the slow activation of the enzyme upon the addition of substrate. Hysteresis is caused by the slow isomerization from an inactive (E*) to the active state. Crystal structures of unliganded, as well as UDP-glucose or UDP-xylose bound hUGDH identified a buried structural element (the T131 loop-α6 helix) as the potential allosteric switch. The allosteric switch connects the active site to hexamer interfaces and adopts a unique conformation depending on the ligation state of the enzyme. Here, we have conducted a series of studies to elucidate the role of the switch in both allostery and hysteresis and established that these phenomena are not only coupled but a feature of many UGDH proteins. Introducing the A136M substitution at the apex of the α6 helix trapped the allosteric switch in the E state. Restricting the movement of the allosteric switch abolished both allostery and hysteresis; providing the first evidence these phenomena are coupled. The movement of the allosteric switch between the E and EΩ states requires a substantial repacking of the protein core This repacking was hypothesized to be facilitated by large packing defects surrounding the allosteric switch. Filling a packing defect that only exists in the E state of hUGDH with the A104L substitution, also abolished both hysteresis and allostery supporting this hypothesis. Analysis of UGDH primary sequences suggested the large to small substitutions that resulted in these packing defects may serve as a motif to predict allostery in UGDH proteins. Caenorhabditis elegans UGDH was identified using this motif, and was shown to be both allosteric and hysteretic, showing the motif can predict allostery in UGDH proteins.
