The molecular architecture of glucose‐1‐phosphate uridylyltransferase

Abstract

Glucose-1-phosphate uridylyltransferase, also referred to as UDP-glucose pyrophosphorylase or UGPase, catalyzes the formation of UDP-glucose from glucose-1-phosphate and UTP. Not surprisingly, given the central role of UDP-glucose in glycogen synthesis and in the production of glycolipids, glycoproteins, and proteoglycans, the enzyme is ubiquitous in nature. Interestingly, however, the prokaryotic and eukaryotic forms of the enzyme are unrelated in amino acid sequence and structure. Here we describe the cloning and structural analysis to 1.9 Å resolution of the UGPase from Escherichia coli. The protein is a tetramer with 222 point group symmetry. Each subunit of the tetramer is dominated by an eight-stranded mixed b-sheet. There are two additional layers of b-sheet (two and three strands) and 10 a-helices. The overall fold of the molecule is remarkably similar to that observed for glucose-1-phosphate thymidylyltransferase in complex with its product, dTDP-glucose. On the basis of this similarity, a UDP-glucose moiety has been positioned into the active site of UGPase. This protein/product model predicts that the side chains of Gln 109 and Asp 137, respectively, serve to anchor the uracil ring and the ribose of UDP-glucose to the protein. The b-phosphoryl group of the product is predicted to lie within hydrogen bonding distance to the e-nitrogen of Lys 202 whereas the carboxylate group of Glu 201 is predicted to bridge the 29-and 39-hydroxyl groups of the glucosyl moiety. Details concerning the overall structure of UGPase and a comparison with glucose-1-phosphate thymidylyltransferase are presented.Keywords: UDP-glucose; glucose-1-phosphate uridylyltransferase; UDP-glucose pyrophosphorylase; X-ray structure; carbohydrates The conversion of b-D-galactose to the more metabolically useful glucose-1-phosphate is accomplished in most organisms by the action of four enzymes that constitute the Leloir pathway (Holden et al. 2003). In the first step of this pathway, b-D-galactose is epimerized to a-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of a-Dgalactose by galactokinase to yield galactose-1-phosphate. Subsequently, in the third step, the enzyme galactose-1-phosphate uridylyltransferase catalyzes the transfer of a UMP group from UDP-glucose to galactose-1-phosphate, thereby generating glucose-1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDPgalactose 4-epimerase. In humans, mutations in the genes that encode the galactokinase, the galactose-1-phosphate uridylyltransferase, or the epimerase can result in the diseased state referred to as galactosemia with clinical manifestations including intellectual retardation, speech disorders, liver dysfunction, and cataract formation. The most common form of galactosemia arises from defects in galactose-1-phosphate uridylyltransferase and as such it has been the subject of intensive kinetic and structural investigations for many years (Holden et al. 2003). Studies have...

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