Abstract
Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.
Highlights
Campylobacter jejuni, a principle cause of acute gastroenteritis, and Helicobacter pylori, a major etiological agent of gastroduodenal disease, are microaerophilic Gram-negative bacteria [1, 2]
Protein Expression and Purification—In this study, C. jejuni proteincoding sequences were cloned from C. jejuni 11168 DNA, and H. pylori sequences were cloned from strain 26695
We have unequivocally demonstrated that the pseudaminic acid (Pse) and 2,4-diacetamido-Bac pathways of C. jejuni and H. pylori are discrete, and we have defined the initial enzymatic steps of both, stemming from the universal substrate UDP-␣-D-GlcNAc
Summary
5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-Lmanno-nonulosonic acid ( known as pseudaminic acid); AltNAc, N-acetyl--L-altrosamine; Bac, 2,4-diamino-2,4,6-trideoxy-␣-D-glucopyranose ( known as bacillosamine); CE, capillary electrophoresis; ␦, chemical shift; HMBC, heteronuclear multiple bond coherence; HSQC, heteronuclear single quantum coherence; J, protoncoupling constant; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; Pgl, general protein glycosylation pathway; PLP, pyridoxal phosphate; TOCSY, total correlation spectroscopy. The flagellins of H. pylori have been shown to be modified with Pse, where glycosylation again appears to be required for assembly of a functional filament [16]. To unequivocally define the Bac and Pse biosynthetic pathways, we have undertaken functional characterization of the C. jejuni and H. pylori flagellar pairs Cj1293/Cj1294 and HP0840/HP0366, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c, using His6-tagged recombinant proteins. These results convincingly demonstrate that the Pse and Bac biosynthetic pathways are discrete, incorporating unique and distinct dehydratase/aminotransferase pairs, and explain the clear phenotypic differences observed between isogenic mutants from the two pathways
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