The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases

Mio, Toshiyuki; Yabe, Tomio; Arisawa, Mikio; Yamada-Okabe, Hisafumi

doi:10.1074/jbc.273.23.14392

Cited by 140 publications

(71 citation statements)

References 29 publications

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“…Dramatic decrease of pyrophosphorylase activity was observed for the G108A. It suggested the Gly 108 in the rGiUAP is a possible catalytic residue, similar to its homolog in ScUAP (12) and EcGlmU (43). In addition, it implied that this catalytic residue for the pyrophosphorylase activity is relatively conserved even the bifunctionality has lost when prokaryotic GlmU evolved into eukaryotic UAP.…”

Section: Discussionmentioning

confidence: 83%

“…Although this motif has been successfully applied to search and identify the UDP-N-acetylglucosamine pyrophosphorylase (UAP) from the yeast data base (12), it cannot be the representative of UAP family. Moreover, the amino acid sequences of prokaryotic UAPs were found to differ significantly from those of eukaryotic UAPs (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Identification of UAP Motifs-An amino acid sequence motif of L(X) 2 GXGTXM(X) 4 PK, where X represents any amino acid was proposed for UAP by comparing the amino acid sequence of the bifunctional N-acetylglucosamine-1-phosphate uridyltransferase from E. coli and UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae and human, respectively (12). Although this motif has been successfully applied to search and identify the UDP-N-acetylglucosamine pyrophosphorylase (UAP) from the yeast data base (12), it cannot be the representative of UAP family.…”

Section: Resultsmentioning

confidence: 99%

“…The activities of these enzymes have been shown to increase from 8-to 4000-fold during encystation of this parasite (5). To date UAP has been purified and characterized from bacteria (11), yeast (12), Neurospora crassa (13), calf liver (11), pig liver (14), and human (12,15,16). However, only the UAP from G. intestinalis has been reported to be developmentally controlled, with 20-fold increase in activity during encystment (5), which makes it distinct from its counterparts in other systems.…”

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confidence: 99%

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

Mok

Edwards

2005

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Mok

Edwards

2005

Journal of Biological Chemistry

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“…The sequence of PsUSP shared low but significant similarity (15%) with UDP-HexNAc pyrophosphorylase (AGX1) (23). Tertiary structure prediction with the three-dimensional-PSSM program showed that the structure of PsUSP is closely related to AGX1 (E-value, 1.44e-62) (29), although GlcNAc 1-phosphate served as a poor substrate for PsUSP.…”

Section: Table V Kinetics Of Udp-sugar Pyrophosphorylasementioning

confidence: 99%

UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts

Kotake

Yamaguchi

Ohzono

et al. 2004

Journal of Biological Chemistry

112

135

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UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDPsugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDPGal, UDP-glucuronic acid, UDP-L-arabinose, and UDPxylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45°C in the presence of 2 mM Mg 2؉ . The apparent K m values for Glc 1-phosphate and L-arabinose 1-phosphate were 0.34 and 0.96 mM, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDPsugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.It is highly probable that the relative amounts and the architecture of cell wall polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are regulated by the level of nucleotide sugars available, as well as by the levels of glycosyltransferases that incorporate monosaccharide units from respective nucleotide sugars into the polymers. Activated nucleotide sugars that serve as glycosyl donors for these polymers in higher plants are generated through de novo pathways, in which various UDP-and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates (1). In the salvage pathways, alternative routes to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of the respective nucleotide triphosphates as co-substrates. Pyrophosphorylases principally catalyze both the following forward (synthesis of NDP-sugars) and reverse (pyrophospholysis) reactions (1): monosaccharides 1-phosphates ϩ NTP % NDP-sugars ϩ PP i , where NTP and NDP are nucleoside triphosphates and diphosphates, respectively. Var...

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Disruption and functional analysis of six ORFs of chromosome IV: YDL103c (QRI1), YDL105w (QRI2), YDL112w (TRM3), YDL113c, YDL116w (NUP84) and YDL167c (NRP1)

Reynaud

Facca

Sor

et al. 2001

Yeast

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We have disrupted six ORFs (YDL103c, YDL105w, YDL112w, YDL113c, YDL116w and YDL167c) located on the left arm of chromosome IV. Except for YDL112w, the short flanking homology strategy was used to construct disruption cassettes using the KanMX4 marker. For YDL112w, a disruption cassette including the LEU2 gene was made. YDL103c and YDL105w are essential genes for vegetative growth. Disruption of YDL112w, YDL113c and YDL167c does not result in any detectable phenotype with the tests we used, while disruption of YDL116w confers slow growth, cryosensitivity and thermosensitivity, and the disrupted diploid homozygotes for the disruption failed to sporulate. Copyright © 2000 John Wiley & Sons, Ltd.

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The Eukaryotic UDP-N-Acetylglucosamine Pyrophosphorylases

Cited by 140 publications

References 29 publications

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

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

UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts

Disruption and functional analysis of six ORFs of chromosome IV: YDL103c (QRI1), YDL105w (QRI2), YDL112w (TRM3), YDL113c, YDL116w (NUP84) and YDL167c (NRP1)

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