A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O1570

Jeffrey S. Rush, Cristina Alaimo, Riccardo Robbiani, Michael Wacker, Charles J. Waechter

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

Escherichia coli strain O157 produces an O-antigen with the repeating tetrasaccharide unit α-D-PerNAc-α-L-Fuc-β-D-Glc-α-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.

Original languageEnglish
Pages (from-to)1671-1680
Number of pages10
JournalJournal of Biological Chemistry
Volume285
Issue number3
DOIs
StatePublished - Jan 15 2010

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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