Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture

The UDP-N-acetylglucosamine pyrophosphorylase in Giardia intestinalis (GiUAP) is one of the five inducible enzymes to synthesize UDP-GalNAc, which is an important precursor for cyst wall synthesis. The recombinant UDP-N-acetylglucosamine pyrophosphorylase (rGiUAP) and its mutants G108A and G210A were expressed and identified by SDS-PAGE, size-exclusion chromatography, Western hybridization, and MALDI mass spectrometry. Sequence comparison with other eukaryotic UAPs has identified three specific motifs. Within these motifs alanine substitution for Gly 108 or Gly 210 dramatically reduced the pyrophosphate synthesis, suggesting these amino acids are catalytic residues. Besides, the rGi-UAP was found to have relaxed binding to other uridine-based nucleotides, suggesting the substrate binding pocket is specific to uridine rather than phosphate group(s). Moreover, thermal denaturation analysis showed a significant increase in T m for the rGiUAP and G108A upon binding of the substrate Mg-UTP. In contrast, G210A showed a decreased T m upon binding of Mg-UTP. These results showed that binding of Mg-UTP increases protein stability of the rGiUAP, and the catalytic residue Gly 210 plays a significant role in stabilizing the protein structure. Such stabilization effect induced by substrate binding might be physiologically important as it favors the production of UDP-GlcNAc and hence the downstream GalNAc, which is crucial to survival of Giardia. These results help to define the essential amino acids for catalysis in the GiUAP and reveal the role of Mg-UTP binding in regulation of protein stability.Giardia intestinalis has long been recognized as one of the most early branching eukaryotes (1). It is also one of the most common causes of gastrointestinal infection in human and other mammals (2). When this parasite travels down the intestine in the host, the transformation from a vegetative trophozoite to an infectious cyst requires the synthesis of a rigid cyst wall to survive the adverse environmental conditions outside the host. As this transformation represents a basic adaptive response of a eukaryote to the environment for propagation, G. intestinalis provides a simple eukaryotic model for differentiation and, the biochemical mechanisms of encystation and the cyst wall synthesis have been the subject of intensive investigations in the past decade.Previous studies have shown that cyst wall contains both carbohydrate and protein components (3-9). One of the major components of the outer cyst wall has been identified as a [D-GalNAc(␤133)-D-GalNAc] n homopolymer by chemical methods, mass spectrometry, and hydrogen nuclear magnetic resonance spectroscopy (10). This GalNAc in G. intestinalis is synthesized from endogenous Glc rather than salvaged from external source (5). The precursor of GalNAc, UDP-GalNAc, is synthesized by five inducible enzymes: glucosamine-6-phosphate isomerase (EC 3.5.99.6); glucosamine-6-phosphate N-acetylase (EC 2.3.1.4); phosphoacetylglucosamine mutase (EC 5.4.2.3, AGM) 2 ; UDP-N-acetylglucosamine py...

“…These motifs were found to surround the substrate binding site in the human UAPs: AGX1 and AGX2 (37) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

Mok

Edwards

2005

“…major USP presents a clear homology with plant USPs and a modest but significant homology with UGPs and UDP-N-acetylglucosamine pyrophosphorylases over the entire sequence. In particular, the pyrophosphorylase glycine-rich consensus motif (25,26) essential for catalysis is highly conserved and additional residues involved in uridine and phosphate binding. As highlighted by STD NMR spectroscopic studies, interactions of the uridine moiety of nucleotide sugars or UTP with LmjUSP are similar to those observed with LmjUGP and play a significant role in substrate binding.…”

Section: Discussionmentioning

confidence: 99%

“…An alignment of L. major USP and UGP is presented in Fig. 2A and highlights the conservation of residues essential for catalytic activity of pyrophosphorylase and for nucleotide sugar binding (25,26). The LmjUSP basic residues Lys-134, His-224, and Lys-434 (corresponding to LmjUGP Lys-95, His-191, and Lys-380) are strictly conserved and predicted to be involved in phosphate binding (26).…”

Section: Identification Of a Putative Udp-sugar Pyrophosphorylase Inmentioning

confidence: 99%

Leishmania UDP-sugar Pyrophosphorylase

Damerow

Lamerz

Haselhorst

et al. 2010

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.

“…Eukaryotic UAPs are divided into two groups, mammalian UAPs and non-mammalian UAPs. The crystal structures of mammalian UAPs have been determined in humans (17) and Mus musculus (Protein Data Bank (PDB) 1VM8). These UAPs consist of three domains, a pyrophosphorylation domain and two other domains whose functions are unknown.…”

mentioning

confidence: 99%

Crystal Structure of Uridine-diphospho-N-acetylglucosamine Pyrophosphorylase from Candida albicans and Catalytic Reaction Mechanism

Maruyama

Nishitani

Nonaka

et al. 2007

Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an S N 2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the ␣-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-GlcNAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the ␣-and ␥-phosphate group of UTP.