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

Namboori, Seema C.; Graham, David E.

doi:10.1016/j.ab.2008.06.034

Cited by 28 publications

(39 citation statements)

References 31 publications

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“…The glycopeptide aa 34 to 47 contains two sequons that are adjacent to one another, and both sites (with the modified asparagines separated by only 2 amino acids) could be found modified with a single GalNAc. The MS data for FlaB2 is consistent with the Western blot results and in vitro studies showing that MMP0352 catalyzes UDP-GlcNAc oxidation at the 3== position (28), thus interfering with the synthesis of the second sugar of the N-linked glycan.…”

Section: Fig 1 Western Blot Detection Of Archaellin Flab2 and Pilin Esupporting

confidence: 75%

“…Attempts to delete either mmp0705 or mmp0706 have been unsuccessful to date (unpublished data), indicating they might play a role in an essential pathway, such as in the synthesis of coenzyme M, a critical coenzyme for methanogenesis (51) that is reported to be modified with ManNAcA (52). The in vitro work of Namboori and Graham on MMP0352 enzymatic activity demonstrated its ability to catalyze the NAD-dependent oxidation of the 3== position of UDP-GlcNAc (28). The genetic and MS data presented in the current work and the role proposed for MMP0352 in the pathway for the generation of the second and ultimately the third sugars of the archaellin N-linked glycan are fully consistent with their findings.…”

Section: Figmentioning

confidence: 99%

“…MMP0350 was also identified as a putative acetyltransferase involved in biosynthesis of the second sugar (27). Additionally, in vitro assays using heterologously expressed proteins have identified other M. maripaludis gene products that are involved in acetamido sugar biosynthesis (28,29), although genetic analysis of these is still lacking. Furthermore, the lipid carrier for the N-glycan in M. maripaludis was identified as a dolichol monophosphate (23).…”

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confidence: 99%

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Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

et al. 2015

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Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-␤-ManNAc3NAmA6Thr-1,4-␤-GlcNAc3NAcA-1,3-␤-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-␣-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the ⌬mmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The ⌬mmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. IMPORTANCEThis work identifies a series of genes in adjacent operons that are shown to encode the enzymes that complete the entire pathway for generation of the second and third sugars of the N-linked tetrasaccharide that modifies archaellins of Methanococcus maripaludis. This posttranslational modification of archaellins is important, as it is necessary for archaellum assembly. Pilins are modified with a different N-glycan consisting of the archaellin tetrasaccharide but with an additional hexose attached to the linking sugar. Mass spectrometry analysis of the pili of one mutant strain provided insight into how this different glycan might ultimately be assembled. This study includes a rare example of an archaeal gene functionally replacing a bacterial gene in a complex sugar biosynthesis pathway. While N-linked glycosylation was initially identified as an exclusively eukaryotic process, it is now well established that this pathway is present in the prokaryotic domains of Archaea and Bacteria as well. The posttranslational modification of proteins with N-linked glycans is believed to be much more widespread in Archaea than in Bacteria. In N-linked glycosylation systems, an oligosaccharyltransferase is required for the transfer of assembled oligosaccharides from the lipid carrier onto target proteins; genes encoding this key signature protein of the pathway have been identified in 166 out of 168 sequ...

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Section: Fig 1 Western Blot Detection Of Archaellin Flab2 and Pilin Esupporting

confidence: 75%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

et al. 2015

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“…In addition, in vitro biochemical work using purified enzymes has further con-tributed to our understanding of the process in this group of Archaea (108)(109)(110).…”

Section: Pathway Of N-linked Glycosylation In Methanogensmentioning

confidence: 99%

“…MMP0353 (a dehydrogenase), MMP0351 (an aminotransferase), MMP0350 (an acetyltransferase), and MMP0357 (an epimerase) share substantial sequence similarity with WpbA, WbpE, WbpD, and WbpI, respectively, while MMP0352 is 48% similar to WbpB albeit with only 37% coverage. A polyhistidine-tagged version of MMP0352 expressed in E. coli has been studied in vitro (109) and was shown to catalyze the oxidation of UDP-GlcNAc to generate the keto sugar UDP-2-acetamido-3-oxo-2,3-dideoxy-␣-D-glucopyranose in a NAD ϩ -dependent fashion. MMP0352 did not oxidize UDP-Glc, UDP-GalNAc, GlcNAc, or glucosamine.…”

Section: Pathway Of N-linked Glycosylation In Methanogensmentioning

confidence: 99%

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

Jarrell

Ding

Meyer

et al. 2014

Microbiol Mol Biol Rev

184

214

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SUMMARY N-glycosylation of proteins is one of the most prevalent posttranslational modifications in nature. Accordingly, a pathway with shared commonalities is found in all three domains of life. While excellent model systems have been developed for studying N-glycosylation in both Eukarya and Bacteria , an understanding of this process in Archaea was hampered until recently by a lack of effective molecular tools. However, within the last decade, impressive advances in the study of the archaeal version of this important pathway have been made for halophiles, methanogens, and thermoacidophiles, combining glycan structural information obtained by mass spectrometry with bioinformatic, genetic, biochemical, and enzymatic data. These studies reveal both features shared with the eukaryal and bacterial domains and novel archaeon-specific aspects. Unique features of N-glycosylation in Archaea include the presence of unusual dolichol lipid carriers, the use of a variety of linking sugars that connect the glycan to proteins, the presence of novel sugars as glycan constituents, the presence of two very different N-linked glycans attached to the same protein, and the ability to vary the N-glycan composition under different growth conditions. These advances are the focus of this review, with an emphasis on N-glycosylation pathways in Haloferax , Methanococcus , and Sulfolobus .

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Ngk1 kinase‐mediated N‐acetylglucosamine metabolism promotes UDP‐GlcNAc biosynthesis in Saccharomyces cerevisiae

Nishikawa,

Karita,

Umekawa

2024

FEBS Letters

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N‐acetylglucosamine (GlcNAc) is an important structural component of the cell wall chitin, N‐glycans, glycolipids, and GPI‐anchors in eukaryotes. GlcNAc kinase phosphorylates GlcNAc into GlcNAc‐6‐phosphate, a precursor of uridine diphosphate N‐acetylglucosamine (UDP‐GlcNAc) that serves as a substrate for glycan synthesis. Although GlcNAc kinase is found widely in organisms ranging from microorganisms to mammals, it has never been found in the model yeast Saccharomyces cerevisiae. Here, we demonstrate the presence of GlcNAc metabolism for UDP‐GlcNAc biosynthesis in S. cerevisiae through Ngk1, a GlcNAc kinase we discovered previously. The overexpression or deletion of Ngk1 in the presence of GlcNAc affected the amount of both UDP‐GlcNAc and chitin, suggesting that GlcNAc metabolism via Ngk1 promotes UDP‐GlcNAc synthesis. Our data suggest that the Ngk1‐mediated GlcNAc metabolism compensates for the hexosamine pathway, a known pathway for UDP‐GlcNAc synthesis.

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

Cited by 28 publications

References 31 publications

Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

Evidence that Biosynthesis of the Second and Third Sugars of the Archaellin Tetrasaccharide in the Archaeon Methanococcus maripaludis Occurs by the Same Pathway Used by Pseudomonas aeruginosa To Make a Di-N-Acetylated Sugar

N-Linked Glycosylation in Archaea: a Structural, Functional, and Genetic Analysis

Ngk1 kinase‐mediated N‐acetylglucosamine metabolism promotes UDP‐GlcNAc biosynthesis in Saccharomyces cerevisiae

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