Enzymes of UDP‐GlcNAc biosynthesis in yeast

Milewski, Sławomir; Gabriel, Iwona; Olchowy, Jarosław

doi:10.1002/yea.1337

Cited by 159 publications

(163 citation statements)

References 101 publications

(136 reference statements)

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“…The phenotypes of the hxk1⌬ and h-d mutants also indicate that GlcNAc does not have to enter the anabolic pathways to induce signaling. Phosphorylation of exogenous GlcNAc by Hxk1 is required to create GlcNAc-6-PO 4 , which is then converted to UDP-GlcNAc for use as a substrate in the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors on proteins (16). Thus, the results indicate that GlcNAc metabolism is not required for it to activate signaling.…”

Section: Nonphosphorylated Glcnac Activates Cell Signaling In Cmentioning

confidence: 94%

“…The genes that mediate GlcNAc catabolism have been identified previously and are known to encode Hxk1, a kinase that phosphorylates GlcNAc to create GlcNAc-6-PO 4 , Dac1, a deacetylase that converts it to glucosamine-6-PO 4 , and Nag1, a deaminase that converts it to fructose-6-PO 4 that can then be used in glycolysis (10,11,15). These experiments also test whether signaling requires the exogenous GlcNAc to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors on proteins (16). Phosphorylation of exogenous GlcNAc by Hxk1 is needed to create GlcNAc-6-PO 4 , which is then converted to UDP-GlcNAc for use as a substrate in the anabolic pathways (16).…”

mentioning

confidence: 99%

“…These experiments also test whether signaling requires the exogenous GlcNAc to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors on proteins (16). Phosphorylation of exogenous GlcNAc by Hxk1 is needed to create GlcNAc-6-PO 4 , which is then converted to UDP-GlcNAc for use as a substrate in the anabolic pathways (16). The results demonstrate that GlcNAc metabolism is not required for induction of hyphal growth or expression of the catabolic genes, thereby identifying a novel role for GlcNAc in cell signaling.…”

mentioning

confidence: 99%

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N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

Naseem¹,

Gunasekera²,

Araya

et al. 2011

Journal of Biological Chemistry

118

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N-Acetylglucosamine (GlcNAc) stimulates important signaling pathways in a wide range of organisms. In the human fungal pathogen Candida albicans, GlcNAc stimulates hyphal cell morphogenesis, virulence genes, and the genes needed to catabolize GlcNAc. Previous studies on the GlcNAc transporter (NGT1) indicated that GlcNAc has to be internalized to induce signaling. Therefore, the role of GlcNAc catabolism was examined by deleting the genes required to phosphorylate, deacetylate, and deaminate GlcNAc to convert it to fructose-6-PO 4 (HXK1, NAG1, and DAC1). As expected, the mutants failed to utilize GlcNAc. Surprisingly, GlcNAc inhibited the growth of the nag1⌬ and dac1⌬ mutants in the presence of other sugars, suggesting that excess GlcNAc-6-PO 4 is deleterious. Interestingly, both hxk1⌬ and an hxk1⌬ nag1⌬ dac1⌬ triple mutant could be efficiently stimulated by GlcNAc to form hyphae. These mutants could also be stimulated to express GlcNAcregulated genes. Because GlcNAc must be phosphorylated by Hxk1 to be catabolized, and also for it to enter the anabolic pathways that form chitin, N-linked glycosylation, and glycosylphosphatidylinositol anchors, the mutant phenotypes indicate that GlcNAc metabolism is not needed to induce signaling in C. albicans. Thus, these studies in C. albicans reveal a novel role for GlcNAc in cell signaling that may also regulate critical pathways in other organisms.

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Section: Nonphosphorylated Glcnac Activates Cell Signaling In Cmentioning

confidence: 94%

mentioning

confidence: 99%

mentioning

confidence: 99%

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N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

Naseem¹,

Gunasekera²,

Araya

et al. 2011

Journal of Biological Chemistry

118

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“…Yeast cells use GlcNAc in N-linked protein glycosylation, in glycosylphosphatidylinositol protein-anchors to the membrane, and in the chitin layer of their cell walls [28]. While S. cerevisiae cell walls have a relatively low chitin content (1-2%), the walls of other fungi contain a high proportion of chitin [29].…”

Section: Measurement Of Udp-glcnac In S Cerevisiaementioning

confidence: 99%

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Namboori

Graham

2008

Analytical Biochemistry

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The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD + -dependent oxidation of the 3″-position of uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations during time series experiments or inhibitor screens.

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“…1B) is responsible for metabolic conversion of fructose-6-phosphate, an intermediate of glycolysis, to UDP-GlcNAc. The highly conserved enzymes in this pathway are essential for yeast viability under normal growth conditions (24). An interruption to the UDP-GlcNAc biosynthetic pathway can be rescued if an appropriate downstream metabolite is delivered into cells.…”

Section: Resultsmentioning

confidence: 99%

Targeted metabolic labeling of yeast N-glycans with unnatural sugars

Breidenbach¹,

Gallagher

King

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridinediphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Δ strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.click chemistry | GlcNAc | metabolic engineering | N-glycosylation | GNA1

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Enzymes of UDP‐GlcNAc biosynthesis in yeast

Cited by 159 publications

References 101 publications

N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Targeted metabolic labeling of yeast N-glycans with unnatural sugars

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