UDP-sugar pyrophosphorylases (UGPs) play a central role in cellular carbohydrate biosynthesis. In mammals, UGP is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. As metabolic switches, UGPs are subject to elaborate regulatory mechanisms. Elucidating these mechanisms is essential for the fundamental understanding of cellular carbohydrate biosynthesis and for the development of new concepts to specifically inhibit individual enzymes. Here we report comprehensive studies of the catalytic mechanism and allosteric regulation of UGPs from the human pathogen Leishmania major and the host using a combination of kinetic X-ray crystallography, biochemical, biophysical and theoretical chemistry methods. The new data on UGP from L. major (LmUGP) filled critical gaps in the knowledge of the UGP mechanism and allowed reconstruction of the complete enzymatic cycle. This study revealed structural changes throughout the cycle, mechanisms of substrate binding, catalysis, product release and allosteric regulation. We show that many of these mechanisms are shared in other nucleotidyltransferases, e.g., in the OAS/cGAS triggers of innate immunity. In contrast to monomeric LmUGP, human enzyme (hUGP) is active as an octamer. We determined the structure of hUGP in complex with its product, which provided first insights into the active site architecture, the substrate binding mode and functionally significant interactions in an octamer. We demonstrate that oligomerization extends the functionality of hUGP in several ways: it (i) facilitates an intermolecular stabilization of the sugar moiety in the active site, (ii) enhances protein stability, and (iii) allows regulation of the hUGP octamer by modification of a single subunit. Due to their central position in carbohydrate biosynthesis, UGPs are attractive targets for drug development against protozoan parasites. Activation mechanisms in LmUGP and hUGP helped us identify an allosteric site on the surface of LmUGP that can be used as a target for developing specific inhibitors. The potential of this site for LmUGP inhibition was experimentally confirmed, and the specific allosteric inhibitor for the pathogen’s enzyme has been developed.

Allostery and catalysis of UDP-sugar pyrophosphorylases suggest a new approach to anti-parasitic treatments

253rd National Meeting (2017)

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