A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

Abstract

In mammals, UDP-glucose pyrophosphorylase (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. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens.

Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium

“…The curve fit gives a Hill coefficient of approximately 1.3, which could indicate very mild positive cooperativity for Gal1P. Wild-type HsUGP, an octameric enzyme, also displays a similar mild cooperativity for its substrate, PP i , in the reverse reaction [24]. However, under the assay conditions employed, we were unable to detect production of UDP-Gal from Gal1P by HsUGP(G116D).…”

“…In the previous study, end-point determinations employing different coupled reactions to detect UDP-Glc or UDP-Gal were used [21], whereas we used a continuous assay based on PPi detection, which is independent of Glc/Gal. Although G116 is located within the nucleotide-binding region of the active site [24], its mutation may affect Glc1P binding indirectly. Since the enzyme follows an ordered sequential Bi-Bi mechanism with UTP as the first substrate, it can be speculated that HsUGP(G116D) binds UTP with nearly wild-type affinity, but its altered orientation within the active site may prevent efficient subsequent binding of Glc1P and in consequence dramatically affect catalysis.…”

“…A previously generated expression plasmid for N-terminally StrepII-tagged HsUGP was used to recombinantly produce the full-length wild-type enzyme as previously described [24]. Using this plasmid as a template, the G116D mutation was introduced employing the same strategy as described earlier [24]. The integrity of the resulting plasmid and the presence of the desired mutation was confirmed by sequencing of the plasmid (GATC Biotech, Konstanz, DE).…”

“…The integrity of the resulting plasmid and the presence of the desired mutation was confirmed by sequencing of the plasmid (GATC Biotech, Konstanz, DE). Recombinant expression in Escherichia coli, affinity chromatographic purification, buffer exchange and storage of both the wild-type and mutant enzyme were likewise carried out as described before, and the purity and integrity of the proteins were confirmed by SDS-PAGE followed by Coomassie protein staining or Western blot analysis [24].…”

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents

“…In order to study the binding mode of GAL-012 and GAL-012-2 to the human GALT, UGP2 and AGX1/UAP1, ligand-protein docking studies were performed as previously using Glide (Schrödinger, LLC) [22]. The X-ray crystal structures of human GALT (PDB ID: 5IN3), UGP2 (PDB ID: 4R7P) [71] and AGX1/UAP1 (PDB ID: 1JVD) [72] were retrieved from PDB, and each protein was prepared by Schrödinger 2015. Grids generation was set up on proteins and saved as job ZIP format.…”