A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157.
Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit alpha-D-PerNAc-alpha-l-Fuc-beta-D-Glc-alpha-D-GalNAc, preassembled on undecaprenyl pyrophosphate (Und-P-P). These studies were conducted to determine whether the biosynthesis of the lipid-linked repeating tetrasaccharide was initiated by the formation of GalNAc-P-P-Und by WecA. When membrane fractions from E. coli strains K12, O157, and PR4019, a WecA-overexpressing strain, were incubated with UDP-[3H]GalNAc, neither the enzymatic synthesis of [3H]GlcNAc-P-P-Und nor [3H]GalNAc-P-P-Und was detected. However, when membrane fractions from strain O157 were incubated with UDP-[3H]GlcNAc, two enzymatically labeled products were observed with the chemical and chromatographic properties of [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und, suggesting that strain O157 contained an epimerase capable of interconverting GlcNAc-P-P-Und and GalNAc-P-P-Und. The presence of a novel epimerase was demonstrated by showing that exogenous [3H]GlcNAc-P-P-Und was converted to [3H]GalNAc-P-P-Und when incubated with membranes from strain O157. When strain O157 was metabolically labeled with [3H]GlcNAc, both [3H]GlcNAc-P-P-Und and [3H]GalNAc-P-P-Und were detected. Transformation of E. coli strain 21546 with the Z3206 gene enabled these cells to synthesize GalNAc-P-P-Und in vivo and in vitro. The reversibility of the epimerase reaction was demonstrated by showing that [3H]GlcNAc-P-P-Und was reformed when membranes from strain O157 were incubated with exogenous [3H]GalNAc-P-P-Und. The inability of Z3206 to complement the loss of the gne gene in the expression of the Campylobacter jejuni N-glycosylation system in E. coli indicated that it does not function as a UDP-GlcNAc/UDP-GalNAc epimerase. Based on these results, GalNAc-P-P-Und is synthesized reversibly by a novel GlcNAc-P-P-Und epimerase after the formation of GlcNAc-P-P-Und by WecA in E. coli O157.
Rush
Jeffrey S
JS
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Alaimo
Cristina
C
Robbiani
Riccardo
R
Wacker
Michael
M
Waechter
Charles J
CJ
D055162:Biocatalysis; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D019453:Escherichia coli O157 / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism
D029968:Escherichia coli Proteins; D011103:Polyisoprenyl Phosphate Monosaccharides; D014542:Uridine Monophosphate; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli; D002238:Carbohydrate Epimerases