Enzymatic Synthesis of UDP-Galactofuranose and an Assay for UDP-Galactopyranose Mutase Based on High-Performance Liquid Chromatography

Lee, Robin; Monsey, D.; Weston, Anthony; Duncan, Kenneth; Rithner, Christopher D.; McNeil, Michael

doi:10.1006/abio.1996.0419

Cited by 73 publications

(55 citation statements)

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“…In Actinobacillus actinomycetemcomitans serotype f, RmlA and RmlB catalyze the formation of dTDP-6-deoxy-D-xylo-hexos-4-ulose, which is converted to dTDP-D-fucopyranose (dTDP-D-Fucp) by dTDP-4-keto-6-deoxy-D-glucose C-4 reductase (Fcd) (27). Synthesis of galactofuranose (Galf) is known to involve mutase action on UDP-galactopyranose (UDP-Galp) (33), and the pathway genes for paratofuranose (Parf) include the genes for a full pathway for CDP-paratopyranose (CDP-Parp) and a mutase gene (43). One might have expected D-Fucf to be synthesized by mutase action on dTDP-D-Fucp.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of the Heteropolysaccharide O Antigen ofEscherichia coliO52 Requires an ABC Transporter: Structural and Genetic Evidence

et al. 2004

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The structural and genetic organization of the Escherichia coli O52 O antigen was studied. As identified by sugar and methylation analysis and nuclear magnetic resonance spectroscopy, the O antigen of E. coli O52 has a partially O-acetylated disaccharide repeating unit (O unit) containing D-fucofuranose and 6-deoxy-D-mannoheptopyranose, as well as a minor 6-deoxy-3-O-methylhexose (most likely, 3-O-methylfucose). The O-antigen gene cluster of E. coli O52, which is located between the galF and gnd genes, was found to contain putative genes for the synthesis of the O-antigen constituents, sugar transferase genes, and ABC-2 transporter genes. Further analysis confirmed that O52 employs an ATP-binding cassette (ABC) transporter-dependent pathway for translocation and polymerization of the O unit. This is the first report of an ABC transporter being involved in translocation of a heteropolysaccharide O antigen in E. coli. Genes specific for E. coli O52 were also identified.The O antigen (O-specific polysaccharide) is a key component of the lipopolysaccharide (LPS) in the outer membrane of many gram-negative bacteria. It consists of repeats of an oligosaccharide unit (O unit), which usually contains two to eight residues of a broad range of both common and rarely occurring sugars and their derivatives. The O antigen is one of the most variable cell constituents, and there are variations in the types of sugars present, their arrangement within the O unit, and the linkages between the O units. The O antigen contributes the major antigenic variability to the cell surface and is subject to intense selection by the host immune system and other environmental factors, such as bacteriophages, which may account for the maintenance of diverse O-antigen forms within species, such as Escherichia coli (42).Three mechanisms for the biosynthesis of O antigen have been recognized, as reviewed by Samuel and Reeves (50). These mechanisms are a Wzy/Wzx-dependent pathway, an ATP-binding cassette (ABC) transporter-dependent pathway, and a synthase-dependent pathway. All three pathways have the same initiation reaction, in which a sugar phosphate is attached to the carrier lipid, undecaprenyl phosphate (Und-P), on the cytosolic face of the plasma membrane. In the Wzy/ Wzx-dependent pathway, additional sugars are added sequentially to form an O unit on the cytosolic face of the plasma membrane. The Und-PP-linked nascent O unit is then transferred across the plasma membrane by the O-unit flippase Wzx and is polymerized on the periplasmic face of the plasma membrane by the O-antigen polymerase Wzy (13). The O antigen is then attached to the independently synthesized lipid A core oligosaccharide to form the LPS (44). This pathway is utilized for the synthesis of the majority of E. coli, Shigella, and Salmonella O antigens studied. In the ABC transporter-dependent pathway, which has been found for only a few E. coli O antigens, additional sugars are added sequentially at the nonreducing end of the growing polymer to form an O antigen on the cyto...

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Section: Resultsmentioning

confidence: 99%

Synthesis of the Heteropolysaccharide O Antigen ofEscherichia coliO52 Requires an ABC Transporter: Structural and Genetic Evidence

et al. 2004

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“…However, in these membranes, UDP-Galf is limiting, so the membranes were augmented with an aliquot of cytoplasmic extract from E. coli CWG288(pWQ66) overexpressing Glf (28). The equilibrium of the Glf-mediated interconversion is such that only about 5% of UDP-Galp is converted into UDP-Galf by the UDP-Galp mutase in the absence of any transferase to draw off the UDP-Galf product (28,30). Supplementation of the WbbO-containing membranes with Glf-containing CWG288 lysate resulted in almost quantitative recovery of a highermolecular-weight lipid intermediate with a migration consistent with a und-PP-linked trisaccharide (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The remaining four genes (wbbM, glf, wbbN, and wbbO) are proposed to be involved in the synthesis of D-galactan I. The glf gene product is a UDPgalactopyranosye mutase, which catalyzes the reversible interconversion of uridine 5Ј-diphospho-␣-D-galactopyranose (UDP-Galp) and uridine 5Ј-diphospho-␣-D-galactofuranose (UDP-Galf) (28,30,36); these two sugar nucleotides provide the precursors for D-galactan I synthesis. The precise roles played by WbbM and WbbN are unknown, but some information is available for WbbO.…”

Section: ␤-D-galf-(133)-␣-d-galp-(13] (D-galactan I) and [33)-␣-dgalpmentioning

confidence: 99%

Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d -Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

2001

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(Fig. 1B). D-Galactan II provides the epitope or epitopes that define the O1 antigen (55). The presence of D-galactan II is required for the resistance of the bacteria to complement-mediated killing in the host, and therefore Klebsiella pneumoniae mutants that produce only D-galactan I are serum sensitive (33).There are two major pathways for the biosynthesis of LPS O-PS. These are designated Wzy dependent and ATP-binding cassette (ABC) transporter dependent (reviewed in references 52 and 53). Both pathways are widely distributed among different genera and are involved in the synthesis of diverse O-PS structures. A third pathway (synthase dependent) is currently confined to a single O-PS (17). In the Wzy-dependent mechanism, undecaprenyl pyrophosphoryl (und-PP)-linked oligosaccharide repeat units are assembled at the cytoplasmic face of the inner membrane. These intermediates are then exported to the periplasmic face, where they provide the direct substrates for polymerization. Export and polymerization require the activity of members of the Wzx and Wzy protein families, respectively. In the ABC transporter-dependent mechanism, the polymer is built by processive transfer of glycosyl residues to an initiating und-PP-linked lipid intermediate at the cytoplasmic face of the inner membrane. After the lipid-linked polymer is synthesized at the cytoplasmic face of the inner membrane, it is exported to the periplasmic face by an ABC transporter (5, 45, 58). The various O-PS biosynthesis pathways are believed to converge with the presence of und-PPlinked polymer at the periplasmic face of the inner membrane, at which point the nascent O-PS is ligated to preformed lipid A core. The completed LPS molecule is then translocated to the outer leaflet of the outer membrane by a process that is still undetermined.In members of the family Enterobacteriaceae, initiation of ABC transporter-dependent

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“…3). We tried to measure the intracellular concentration of UDP-glucuronic acid in the wild-type and mutated C. neoformans cells by a high-pressure liquid chromatography procedure (27). However, the wild-type concentration was at the limit of detection of the system and we did not obtain conclusive results (data not shown).…”

Section: Identification Of Ugd1mentioning

confidence: 96%

UGD1 , Encoding the Cryptococcus neoformans UDP-Glucose Dehydrogenase, Is Essential for Growth at 37°C and for Capsule Biosynthesis

Moyrand

Janbon

2004

Eukaryot Cell

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We report the identification and disruption of the Cryptococcus neoformans var. grubii UGD1 gene encoding the UDP-glucose dehydrogenase, which catalyzes the conversion of UDP-glucose into UDP-glucuronic acid. Deletion of UGD1 led to modifications in the cell wall, as revealed by changes in the sensitivity of ugd1⌬ cells to sodium dodecyl sulfate, NaCl, and sorbitol. Moreover, two of the yeast's major virulence factors-capsule biosynthesis and the ability to grow at 37°C-were impaired in ugd1⌬ strains. These results suggest that the UDP-dehydrogenase represents the major, and maybe only, biosynthetic pathway for UDP-glucuronic acid in C. neoformans. Consequently, deletion of UGD1 blocked not only the synthesis of UDP-glucuronic acid but also that of UDP-xylose. To differentiate the phenotype(s) associated with the UDP-glucuronic acid defect alone from those linked to the UDP-xylose defect, ugd1⌬ mutants were phenotypically compared to strains from which the gene encoding UDP-xylose synthase (i.e., that required for synthesis of UDP-xylose) had been deleted. Finally, studies of strains from which one of the four CAP genes (CAP10, CAP59, CAP60, or CAP64) had been deleted revealed common cell wall phenotypes associated with the acapsular state.

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Enzymatic Synthesis of UDP-Galactofuranose and an Assay for UDP-Galactopyranose Mutase Based on High-Performance Liquid Chromatography

Cited by 73 publications

References 1 publication

Synthesis of the Heteropolysaccharide O Antigen ofEscherichia coliO52 Requires an ABC Transporter: Structural and Genetic Evidence

Synthesis of the Heteropolysaccharide O Antigen ofEscherichia coliO52 Requires an ABC Transporter: Structural and Genetic Evidence

Functional Analysis of the Galactosyltransferases Required for Biosynthesis of d -Galactan I, a Component of the Lipopolysaccharide O1 Antigen of Klebsiella pneumoniae

UGD1 , Encoding the Cryptococcus neoformans UDP-Glucose Dehydrogenase, Is Essential for Growth at 37°C and for Capsule Biosynthesis

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