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J. Biol. Chem., Vol. 281, Issue 2, 723-732, January 13, 2006

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Functional Characterization of Dehydratase/Aminotransferase Pairs from Helicobacter and Campylobacter

ENZYMES DISTINGUISHING THE PSEUDAMINIC ACID AND BACILLOSAMINE BIOSYNTHETIC PATHWAYS^*

From the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A OR6, Canada

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--D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP--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 His₆-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--L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy--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--D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy--L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy--D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy--D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy--D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy--D-glucopyranose.

Received for publication, October 11, 2005 , and in revised form, November 10, 2005.

^* This study was supported by the National Research Council Genomics and Health Initiative. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and supplemental Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 613-990-0839; Fax: 613-952-9092; E-mail: susan.logan{at}nrc-cnrc.gc.ca.

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