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Glycobiology Advance Access originally published online on June 3, 2006
Glycobiology 2006 16(9):8C-14C; doi:10.1093/glycob/cwl010
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

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Elucidation of the CMP-pseudaminic acid pathway in Helicobacter pylori: synthesis from UDP-N-acetylglucosamine by a single enzymatic reaction

Ian C. Schoenhofen, David J. McNally, Jean-Robert Brisson and Susan M. Logan1

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

1 To whom correspondence should be addressed; e-mail: susan.logan{at}nrc-cnrc.gc.ca

Received on May 5, 2006; accepted on May 31, 2006

Flagellin glycosylation is a necessary modification allowing flagellar assembly, bacterial motility, colonization, and hence virulence for the gastrointestinal pathogen Helicobacter pylori [Josenhans, C., Vossebein, L., Friedrich, S., and Suerbaum, S. (2002) FEMS Microbiol. Lett., 210, 165–172; Schirm, M., Schoenhofen, I.C., Logan, S.M., Waldron, K.C., and Thibault, P. (2005) Anal. Chem., 77, 7774–7782]. A causative agent of gastric and duodenal ulcers, H. pylori, heavily modifies its flagellin with the sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-{alpha}-L-manno-nonulosonic acid (pseudaminic acid). Because this sugar is unique to bacteria, its biosynthetic pathway offers potential as a novel therapeutic target. We have identified six H. pylori enzymes, which reconstitute the complete biosynthesis of pseudaminic acid, and its nucleotide-activated form CMP-pseudaminic acid, from UDP-N-acetylglucosamine (UDP-GlcNAc). The pathway intermediates and final product were identified from monitoring sequential reactions with nuclear magnetic resonance (NMR) spectroscopy, thereby confirming the function of each biosynthetic enzyme. Remarkably, the conversion of UDP-GlcNAc to CMP-pseudaminic acid was achieved in a single reaction combining six enzymes. This represents the first complete in vitro enzymatic synthesis of a sialic acid-like sugar and sets the groundwork for future small molecule inhibitor screening and design. Moreover, this study provides a strategy for efficient large-scale synthesis of novel medically relevant bacterial sugars that has not been attainable by chemical methods alone.

Key words: biosynthetic pathway elucidation / CMP-pseudaminic acid / enzymatic synthesis / flagellin glycosylation / sialic acid

aminotransferase; PseH, N-acetyltransferase; PseG, nucleotidase; PseI, pseudaminic acid synthase; PseF, CMP-pseudaminic acid synthetase; and (I) UDP-GlcNAc; (II) UDP-2-acetamido-2,6-dideoxy-b-l-arabino-hexos-4-ulose; (III) UDP-4-amino-4,6-dideoxy-b-l-AltNAc; (IV) UDP-2,4-diacetamido-2,4,6-trideoxy-b-l-altropyranose; (V) 2,4-diacetamido-2,4,6-trideoxy-b-l-altropyranose; (VI) pseudaminic acid; (VII) CMP-pseudaminic acid. The assignment of roman numerals to each compound is consistent with label designations found throughout the text. Pyranose rings are shown as their predominant chair conformation in solution determined from nuclear Overhauser effects (NOEs) and JH,H coupling constants.


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