Leishmania UDP-sugar Pyrophosphorylase

Abstract

The Leishmania parasite glycocalyx is rich in galactosecontaining glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

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

“…The commercially available EnzChek pyrophosphate assay (life technologies), based on detection of PP i , was used to continuously monitor the hUGP forward reaction at fixed substrate concentrations of 1 mM UTP and 2 mM Glc-1-P. For the reverse UGP reaction, the formation of UTP was monitored using a continuous enzymatic assay utilizing CTP-synthase as described previously7 at fixed substrate concentrations of 1 mM UDP-Glc and 2 mM PP i under the aforementioned buffer conditions. Enzymatic reactions were performed in 100 μl volumes in 96-well half-area flat-bottom microplates (Greiner Bio-One) and initiated by the addition of recombinant hUGP in suitable dilution.…”

“…UGP follows an ordered sequential Bi Bi mechanism in both directions2 and uses Mg 2+ as an essential cofactor3. With the exception of plants and certain protozoa, where a second enzyme with broader substrate specificity can form UDP-Glc4567, UGP is the only enzyme capable of producing UDP-Glc from glucose-1-phosphate (Glc-1-P) and uridine triphosphate (UTP). Consistent with its vital role, no eukaryotic UGP loss-of-function mutants are known to occur naturally, and the only mammalian model system of impaired UGP function is a hamster fibroblast cell line in which a point mutation in the UGP gene causes a dramatic reduction of UDP-Glc levels8.…”

Recent advances in the enzymatic synthesis of sugar-nucleotides using nucleotidylyltransferases and glycosyltransferases

“…New enzymes are also emerging, typified by a recent report from Routier et al who described the characterisation of a new bacterial UDP-sugar pyrophosphorylase from Leishmania. 38 The enzyme displayed a close relation to plant UDP-sugar pyrophosphorylases, capable of providing UDP-Gal without the Leloir pathway dependence of UDP-Glc and was able to process Gal-and Glc 1-phosphates along with other hexose and pentose 1-phosphates, but notably not hexosamine 1-phosphates.…”

Characterization, Localization, Essentiality, and High-Resolution Crystal Structure of Glucosamine 6-Phosphate N -Acetyltransferase from Trypanosoma brucei

“…However, because almost half of the genes in T. brucei encode hypothetical proteins of unknown function, that fewer still are experimentally annotated, and that the GNAT enzyme superfamily is one of the largest known, the possibility of redundancy certainly could not be excluded prior to experimental verification. A recent and relevant case in point is the unexpected nonessentiality of UDP-Glc pyrophosphorylase in L. major, where it transpires that its function can be compensated for by a relatively nonspecific UDP-sugar pyrophosphorylase (8) that is also present in T. cruzi (57).…”