Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

Westman, Erin L.; McNally, David J.; Charchoglyan, Armen; Brewer, Dyanne; Field, Robert A.; Lam, Joseph S.

doi:10.1074/jbc.m808583200

Cited by 25 publications

(26 citation statements)

References 25 publications

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“…After the review of this report, a preliminary study from the Lam group on this biosynthetic pathway appeared in press. This study described the required coupling of WbpB and WbpE for function; however, it unfortunately provides no information about cofactor requirements and failed to observe the α-KG-dependent dehydrogenase activity of WbpB in the absence of WbpE, instead attributing the pairing of WbpB and WbpE to a necessary physical interaction alone (44). Investigations are currently underway in our laboratory to further characterize the coupling of WbpB and WbpE as well as the potential applications of the UDP-GlcNAc(3keto)A intermediate.…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in Pseudomonas aeruginosa PAO1

Larkin¹,

Imperiali²

2009

Biochemistry

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The B-band O-antigen of the lipopolysaccharide found in the opportunistic pathogen Pseudomonas aeruginosa PAO1 (serotype O5) comprises a repeating trisaccharide unit that is critical for virulence and protection from host defense systems. One of the carbohydrates in this repeating unit, the rare diacetylated aminuronic acid derivative 2,3-diacetamido-2,3-dideoxy-β-D-mannuronic acid (ManNAc(3NAc)A), is thought to be produced by five enzymes (WbpA, WbpB, WbpE, WbpD and WbpI) in a stepwise manner starting from UDP-GlcNAc. Although the genes responsible for the biosynthesis of this sugar are known, only two of the five encoded proteins (WbpA and WbpI) have been thoroughly investigated. In this report, we describe the cloning, overexpression, purification and biochemical characterization of the three central enzymes in this pathway, WbpB, WbpE, and WbpD. Using a combination of capillary electrophoresis, RP-HPLC and NMR spectroscopy, we show that WbpB and WbpE are a dehydrogenase/aminotransferase pair that converts UDP-GlcNAcA to UDP-GlcNAc(3NH 2 )A in a coupled reaction via a unique NAD + recycling pathway. In addition, we confirm that WbpD catalyzes the acetylation of UDP-GlcNAc(3NH 2 )A to give UDP-GlcNAc (3NAc)A. Notably, WbpA, WbpB, WbpE, WbpD and WbpI can be combined in vitro to generate UDP-ManNAc(3NAc)A in a single reaction vessel, thereby providing supplies of this complex glycosyl donor for future studies of LPS assembly. This work completes the biochemical characterization of the enzymes in this pathway and provides novel targets for potential therapeutics to combat infections with drug resistant P. aeruginosa strains.The gram-negative pathogen Pseudomonas aeruginosa is a versatile organism responsible for infection in immunocompromised individuals (1). It is a major source of hospital-acquired pneumonia and bacteremia, causes severe inflammation and pulmonary failure in cystic fibrosis patients, and has emerged as a serious public health threat (2-5). Effective treatment of P. aeruginosa infection has proved challenging due to the strong inherent resistance of the organism to traditional antibiotics and the increasing emergence of multi-drug resistant strains (6-8). While several vaccines for P. aeruginosa have been described, none have thus far achieved clinical success (9). † This work was supported by a grant from National Institutes of Health (GM039334 to B.I.). *To whom correspondence should be addressed: Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-1838; Fax: (617) 452-2419; Email: E-mail: imper@mit.edu. Table of constructs and oligonucleotides used in this study, as well as full 1 H, 13 C and COSY NMR spectra of UDP-GlcNAc(3NH 2 )A and UDP-GlcNAc(3NAc)A. This material is available free of charge via the Internet at http://pubs.acs.org. SUPPORTING INFORMATION AVAILABLE NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 16. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manusc...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in Pseudomonas aeruginosa PAO1

Larkin¹,

Imperiali²

2009

Biochemistry

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“…We also present evidence that biosynthesis of UDPManNAc3NAcA occurs in a manner similar to the WbpABEDI pathway that generates UDP-ManNAc3NAcA as part of O-specific antigen (OSA) biosynthesis in Pseudomonas aeruginosa PAO1 lipopolysaccharide (30,31).…”

mentioning

confidence: 87%

“…MMP0350, MMP0351, MMP0352, MMP0353, and MMP0357 were found to have high sequence similarity to the WbpD, WbpE, WbpB, WbpA, and WbpI Pseudomonas aeruginosa proteins, respectively. In P. aeruginosa PAO1, these five gene products are known to convert UDP-GlcNAc to UDP-ManNAc3NAcA in a stepwise fashion as part of O5-specific antigen biosynthesis (30,31,44).…”

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

confidence: 99%

“…ManNAc3NAcA is a component of OSA of O5-serotype P. aeruginosa PAO1; loss of UDP-ManNAc3NAcA biosynthesis results in loss of OSA expression (30,31). ManNAc3NAcA is also likely a direct precursor of the third sugar of the M. maripaludis archaellin glycan and could then undergo further modifications by AglXYZ and AglU to generate ManNAc3NAmA6Thr.…”

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

confidence: 99%

“…In P. aeruginosa, the conversion of UDPGlcNAc to UDP-ManNAc3NAcA requires the sequential enzy-matic activities of WbpA, WbpB, WbpE, WbpD, and WbpI. Inframe deletions and subsequent complementation studies have shown that all steps are essential for the synthesis of UDPManNAc3NAcA and that without this pathway the O5 OSA is not made (30,31). Biosynthesis of UDP-ManNAc3NAcA in Bordetella pertussis proceeds by the identical scheme, and successful crosscomplementation of P. aeruginosa wbp deletion strains with the corresponding B. pertussis gene has been demonstrated (47).…”

Section: Figmentioning

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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Acquisition of Resistance to PEGylated Branched Polyethylenimine Increases Pseudomonas Aeruginosa Susceptibility to Aminoglycosides

Best,

Ferrell,

Boris

et al. 2024

ChemMedChem

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PEGylated branched polyethylenimine (PEG‐BPEI) has antibacterial and antibiofilm properties. Exposure to PEG‐BPEI through serial passage leads to resistant P. aeruginosa strains. The minimum inhibitory concentration (MIC) of 600 Da BPEI and PEGylated 600 Da BPEI (PEG‐BPEI) in the wild‐type PAO1 strain is 16 µg/ml while, after 15 serial passages, the MIC increased to 1024 µg/mL. An additional 15 rounds of passage in the absence of BPEI or PEG‐BPEI did not change the 1024 µg/mL MIC. Gentamicin, Neomycin, and Tobramycin, cationic antibiotics that inhibit protein synthesis, have a 16‐32 fold reduction of MIC values in PEG350‐BPEI resistant strains, suggesting increased permeation. The influx of these antibiotics occurs using a self‐mediated uptake mechanism, perhaps due to changes in the outer membrane Data show that resistance causes changes in genes related to outer membrane lipopolysaccharide (LPS) assembly. Mutations were noted in the gene coding for the polymerase Wzy that participates in the assembly of the O‐antigen region. Other mutations were noted with wbpE and wbpI of the Wbp pathway responsible for the enzymatic synthesis of ManNAc(3NAc)A in the LPS of P. aeruginosa. These changes suggest that PEG‐BPEI resistance can be countered with antibiotics to prevent the emergence of PEG‐BPEI resistant bacterial populations.

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Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

Cited by 25 publications

References 25 publications

Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in Pseudomonas aeruginosa PAO1

Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in Pseudomonas aeruginosa PAO1

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

Acquisition of Resistance to PEGylated Branched Polyethylenimine Increases Pseudomonas Aeruginosa Susceptibility to Aminoglycosides

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