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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-beta-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 lipopolysaccharide 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.

Titel:	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.
Autor/in / Beteiligte Person:	Larkin, A ; Imperiali, B
Zeitschrift:	Biochemistry, Jg. 48 (2009-06-16), Heft 23, S. 5446-55
Veröffentlichung:	Washington, American Chemical Society., 2009
Medientyp:	academicJournal
ISSN:	1520-4995 (electronic)
DOI:	10.1021/bi900186u
Schlagwort:	Bacterial Proteins chemistry Bacterial Proteins genetics Carbohydrate Sequence Electrophoresis, Capillary Lipopolysaccharides chemistry Lipopolysaccharides metabolism Molecular Sequence Data Multigene Family Pseudomonas aeruginosa metabolism Signal Transduction Uridine Diphosphate Sugars metabolism Uronic Acids chemistry Bacterial Proteins metabolism O Antigens metabolism Pseudomonas aeruginosa enzymology Uronic Acids metabolism
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article; Research Support, N.I.H., Extramural Language: English [Biochemistry] 2009 Jun 16; Vol. 48 (23), pp. 5446-55. MeSH Terms: Bacterial Proteins / metabolism ; O Antigens / metabolism ; Pseudomonas aeruginosa / enzymology ; Uronic Acids / metabolism ; Bacterial Proteins / chemistry ; Bacterial Proteins / genetics ; Carbohydrate Sequence ; Electrophoresis, Capillary ; Lipopolysaccharides / chemistry ; Lipopolysaccharides / metabolism ; Molecular Sequence Data ; Multigene Family ; Pseudomonas aeruginosa / metabolism ; Signal Transduction ; Uridine Diphosphate Sugars / metabolism ; Uronic Acids / chemistry References: FEBS Lett. 1994 Jan 17;337(3):289-92. (PMID: 8293817) ; Clin Microbiol Infect. 2007 Jun;13(6):560-78. (PMID: 17266725) ; Infect Immun. 1984 May;44(2):508-13. (PMID: 6425224) ; Mol Microbiol. 1996 Jan;19(1):37-52. (PMID: 8821935) ; Infect Immun. 1994 Mar;62(3):809-17. (PMID: 8112851) ; Infect Immun. 1996 Jan;64(1):37-43. (PMID: 8557368) ; FEMS Immunol Med Microbiol. 2006 Feb;46(1):85-99. (PMID: 16420601) ; J Bacteriol. 1996 Jan;178(1):232-9. (PMID: 8550422) ; J Clin Microbiol. 1989 May;27(5):962-7. (PMID: 2501356) ; Int J Med Microbiol. 2007 Sep;297(5):277-95. (PMID: 17466590) ; Vaccine. 2008 Feb 20;26(8):1011-24. (PMID: 18242792) ; J Biol Chem. 2008 Oct 10;283(41):27937-27946. (PMID: 18667421) ; Mol Microbiol. 2005 Sep;57(5):1288-303. (PMID: 16102001) ; J Biol Chem. 2006 Sep 22;281(38):27733-43. (PMID: 16690622) ; J Bacteriol. 2008 Sep;190(18):6060-9. (PMID: 18621892) ; Carbohydr Res. 2007 Feb 26;342(3-4):460-6. (PMID: 17087923) ; Clin Infect Dis. 1998 Aug;27 Suppl 1:S93-9. (PMID: 9710677) ; Rev Infect Dis. 1985 Nov-Dec;7 Suppl 4:S778-82. (PMID: 3909337) ; Science. 1995 Nov 10;270(5238):997-1000. (PMID: 7481807) ; Biochemistry. 2006 Nov 14;45(45):13659-69. (PMID: 17087520) ; Microbiol Mol Biol Rev. 1999 Sep;63(3):523-53. (PMID: 10477307) ; J Biol Chem. 2004 Sep 3;279(36):37551-8. (PMID: 15226302) ; Eur J Biochem. 1989 Nov 20;185(3):615-9. (PMID: 2574107) ; J Biol Chem. 1951 Mar;189(1):109-14. (PMID: 14832222) ; Carbohydr Res. 2009 Mar 31;344(5):648-53. (PMID: 19203750) ; PLoS Pathog. 2008 Feb 8;4(2):e43. (PMID: 18282104) ; Biochem J. 2007 Jul 1;405(1):123-30. (PMID: 17346239) ; J Biol Chem. 2005 Apr 29;280(17):16586-93. (PMID: 15723834) ; J Biol Chem. 2005 May 27;280(21):20902-8. (PMID: 15790564) ; J Endotoxin Res. 2006;12(6):324-36. (PMID: 17254386) ; FEBS Lett. 2000 Jul 14;477(1-2):8-14. (PMID: 10899302) ; Biochem J. 1986 Nov 1;239(3):733-8. (PMID: 3030271) ; Infect Immun. 2008 Oct;76(10):4414-21. (PMID: 18663007) ; Protein Sci. 2007 Jun;16(6):1230-5. (PMID: 17473010) ; Biokhimiia. 1994 Dec;59(12):1784-851. (PMID: 7533007) ; FEMS Microbiol Lett. 2000 Aug 15;189(2):135-41. (PMID: 10930727) ; J Biol Chem. 2001 Jul 20;276(29):27329-34. (PMID: 11313358) ; J Bacteriol. 2008 Jun;190(11):3793-8. (PMID: 18390652) ; Microbiology (Reading). 1999 Jul;145 ( Pt 7):1575-1583. (PMID: 10439396) ; J Biol Chem. 2006 Jan 13;281(2):723-32. (PMID: 16286454) ; J Biol Chem. 2004 Jun 11;279(24):25400-10. (PMID: 15044494) ; Infect Immun. 2007 Nov;75(11):5313-24. (PMID: 17724070) ; J Biol Chem. 2009 May 1;284(18):11854-62. (PMID: 19282284) ; Mol Microbiol. 1996 Nov;22(3):481-95. (PMID: 8939432) ; Acta Microbiol Hung. 1988;35(1):3-24. (PMID: 3134788) ; Infect Immun. 1983 Oct;42(1):170-7. (PMID: 6413410) Grant Information: R01 GM039334 United States GM NIGMS NIH HHS; R01 GM039334-22 United States GM NIGMS NIH HHS; GM039334 United States GM NIGMS NIH HHS Substance Nomenclature: 0 (Bacterial Proteins) ; 0 (Lipopolysaccharides) ; 0 (O Antigens) ; 0 (Uridine Diphosphate Sugars) ; 0 (Uronic Acids) ; 21654-37-5 (UDP-N-acetylglucosaminuronic acid) ; 82953-40-0 (2,3-diacetamido-2,3-dideoxymannuronic acid) Entry Date(s): Date Created: 20090408 Date Completed: 20090730 Latest Revision: 20211020 Update Code: 20240513 PubMed Central ID: PMC2694220

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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.