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PMID:20595390
| Citation |
Pinta, E, Duda, KA, Hanuszkiewicz, A, Salminen, TA, Bengoechea, JA, Hyytiäinen, H, Lindner, B, Radziejewska-Lebrecht, J, Holst, O and Skurnik, M (2010) Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core. J. Biol. Chem. 285:28333-42 |
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| Abstract |
Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities. |
| Links |
PubMed PMC2934697 Online version:10.1074/jbc.M110.111336 |
| Keywords |
Antibodies, Monoclonal/metabolism; Bacteriophages/metabolism; Catalytic Domain; Computational Biology; Drug Resistance, Bacterial; Galactose/chemistry; Galactose/metabolism; Glycosyltransferases/chemistry; Glycosyltransferases/genetics; Glycosyltransferases/metabolism; Lipopolysaccharides/biosynthesis; Lipopolysaccharides/chemistry; Models, Molecular; Multigene Family; Mutagenesis, Site-Directed; O Antigens/chemistry; O Antigens/metabolism; Oligosaccharides/chemistry; Oligosaccharides/metabolism; Polymyxin B/pharmacology; Yersinia enterocolitica/drug effects; Yersinia enterocolitica/enzymology; Yersinia enterocolitica/genetics; Yersinia enterocolitica/metabolism |
| edit table |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0047270: lipopolysaccharide glucosyltransferase II activity |
ECO:0000315: |
F |
Table 4, S2, and S3. Mutant lacks terminal glucose residue on LOS chain. |
complete | ||||
|
enables |
GO:0047270: lipopolysaccharide glucosyltransferase II activity |
ECO:0000315: mutant phenotype evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0008919: lipopolysaccharide glucosyltransferase I activity |
ECO:0000315: |
F |
Table 4, S2, S3. MALDI MS of mutant products shows lack of glucose side chain from LOS chain. |
complete | ||||
|
enables |
GO:0008919: lipopolysaccharide glucosyltransferase I activity |
ECO:0000315: mutant phenotype evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0008376: acetylgalactosaminyltransferase activity |
ECO:0000315: |
F |
Table 4, S2, and S3. Mutant terminates before GalNAc residue on LOS chain. |
complete | ||||
|
enables |
GO:0008376: acetylgalactosaminyltransferase activity |
ECO:0000315: mutant phenotype evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
| GO:0035250 : UDP-galactosyltransferase activity |
ECO:0000315: |
F |
Fig 1, Table 4. Chemical analysis of YeO3-c-wbcN1 mutant revealed truncated OC structure containing only Sugp and GalNAc residues. WT Chemotype of OC was restored by complementation with WT wbcN. |
complete | ||||
|
enables |
GO:0035250: UDP-galactosyltransferase activity |
ECO:0000315: mutant phenotype evidence used in manual assertion |
F |
Seeded From UniProt |
complete | |||
See also
References
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