Isolation and identification of uridine(5′)-diphospho(1)-2,3-diacetamido-2,3-dideoxy-α-<scp>d</scp>-glucopyra nuronic acid from <i>Pseudomonas aeruginosa</i> P1-III

Okuda, Satoshi; Murata, Shizuaki; Suzuki, Naomichi

doi:10.1042/bj2390733

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…ManNAc(3NAc)A, the C2-epimer of GlcNAc(3NAc)A, is present not only in the LPS of P. aeruginosa and B. pertussis , but also in the cell wall polysaccharide of the Gram-positive thermophile, Bacillus stearothermophilus 41 . GlcNAc(3NAc)A has been reported to be present in the LPS of a number of P. aeruginosa strains, including P1-III and P14, the N -linked glycans of the methanogenic archaea, Methanococcus voltae and Methanococcus maripalidus , and the cell surface polysaccharide of the Gram-negative spirochete, Treponema medium 42 43 44 45 . A partial genome research survey revealed that Thermus thermophiles ( Deinococcus-Thermus ), Zobellia galactanivorans ( Bacteroidetes ), Wolinella succinogenes ( Epsilonproteobacteria ) and Photorhabdus asymbiotica ( Gammaproteobacteria ) possess 5 genes in the Wbp pathway.…”

Section: Discussionmentioning

confidence: 99%

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

Shoji

Satô

Yukitake

et al. 2014

Sci Rep

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The periodontal pathogen Porphyromonas gingivalis has two different lipopolysaccharide (LPS) molecules, O-LPS and A-LPS. We have recently shown that P. gingivalis strain HG66 lacks A-LPS. Here, we found that introduction of a wild-type wbpB gene into strain HG66 restored formation of A-LPS. Sequencing of the wbpB gene from strain HG66 revealed the presence of a nonsense mutation in the gene. The wbpB gene product is a member of the Wbp pathway, which plays a role in the synthesis of UDP-ManNAc(3NAc)A in Pseudomonas aeruginosa; UDP-ManNAc(3NAc)A is sequentially synthesized by the WbpA, WbpB, WbpE, WbpD and WbpI proteins. We then determined the effect of the PGN_0002 gene, a wbpD homolog, on the biosynthesis of A-LPS. A PGN_0002-deficient mutant demonstrated an A-LPS biosynthesis deficiency. Taken together with previous studies, the present results suggest that the final product synthesized by the Wbp pathway is one of the sugar substrates necessary for the biosynthesis of A-LPS.

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Section: Discussionmentioning

confidence: 99%

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

Shoji

Satô

Yukitake

et al. 2014

Sci Rep

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“…These distinctive molecules have primarily been identified in the complex cell wall matrices of pathogenic bacteria and the glycoproteins of certain archaea. To date, GlcNAc(3NAc)A has been detected in the LPS of a number of P. aeruginosa strains, including P1−III and P14, and the unique N -linked glycans of the methanogenic archaea Methanococcus voltae and Methanococcus maripalidus − . In addition to P. aeruginosa PAO1 (serotype O5), ManNAc(3NAc)A, the C2 epimer of GlcNAc(3NAc)A, is present in the LPS of other P. aeruginosa strains, as well as in the cell wall polysaccharide of the Gram-positive thermophile Bacillus stearothermophilus and the pathogens Bordatella pertussis , Bordatella parapertussis , and Bordatella bronchiseptica − .…”

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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“…The 13 C data for UDP-GlcNAc at 10 mM (Table III) were obtained both directly by recording the one-dimensional 1 H -decoupled 13 C NMR spectrum and indirectly by the 1 Hdetected two-dimensional NMR analysis (data not shown). As C spectrum at high digitization.…”

Section: Evaluation Of the Structure Of Compound A By One-dimensionalmentioning

confidence: 99%

“…For instance, Okuda et al (13) have described the isolation and characterization of UDP-2,3-diacetamido-2,3-dideoxy-D-glucuronic acid from Pseudomonas aeruginosa P1-III, but its enzymatic synthesis was not studied. He and Liu (14) have described genes and enzymes for the biosynthesis of various 3-amino-3-deoxy-sugars.…”

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confidence: 99%

Oxidation and Transamination of the 3″-Position of UDP-N-Acetylglucosamine by Enzymes from Acidithiobacillus ferrooxidans

Sweet

Ribeiro

Raetz

2004

Journal of Biological Chemistry

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Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a ␤-1,6-linked glucosamine disaccharide backbone. However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-D-glucopyranose. We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino-2,3-dideoxy-D-glucopyranose moiety. The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A. ferrooxidans. Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD ؉ -dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP 2-acetamido-3-amino-2,3-dideoxy-␣-D-glucopyranose (UDP-GlcNAc3N). Both gnnA and gnnB were cloned and expressed in E. coli using pET23c؉. In the presence of L-glutamate and NAD ؉ , both proteins were required for the conversion of [␣-32 P]UDPGlcNAc to a novel, less negatively charged sugar nucleotide shown to be [␣-32 P]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, ϳ0.4 mg of the presumptive UDP-GlcNAc3N was synthesized. The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH 2 . As shown in the accompanying papers (Sweet, C.

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Isolation and identification of uridine(5′)-diphospho(1)-2,3-diacetamido-2,3-dideoxy-α-d-glucopyra nuronic acid from Pseudomonas aeruginosa P1-III

Cited by 8 publications

References 25 publications

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

Involvement of the Wbp pathway in the biosynthesis of Porphyromonas gingivalis lipopolysaccharide with anionic polysaccharide

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

Oxidation and Transamination of the 3″-Position of UDP-N-Acetylglucosamine by Enzymes from Acidithiobacillus ferrooxidans

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