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Glycobiology Advance Access originally published online on April 6, 2005
Glycobiology 2005 15(7):721-733; doi:10.1093/glycob/cwi057
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org

Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori

Susan M. Logan1, Eleonora Altman, Oksana Mykytczuk, Jean-Robert Brisson, Vandana Chandan, Frank St. Michael, Amara Masson, Sonia Leclerc, Koji Hiratsuka, Natalia Smirnova, Jianjun Li, Yuyang Wu and Warren W. Wakarchuk

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 February 2, 2005; revised on March 9, 2005; accepted on March 18, 2005

Activity screening and insertional inactivation of lipopolysaccharide (LPS) biosynthetic genes in Helicobacter pylori have led to the successful characterization of two key enzymes encoded by HP0159 (JHP0147) and HP1105 (JHP1032) open reading frames (ORFs) which are members of the large and diverse carbohydrate active enzymes (CAZY) GT-8 (rfaJ) family of glycosyltransferases. Activity screening of a genomic library led to the identification of the enzyme involved in the biosynthesis of the type 2 N-acetyl-lactosamine O-chain backbone, the ß-1,3-N-acetyl-glucosaminyl transferase. In addition, the activity screening approach led to the identification and characterization of a key core biosynthetic enzyme responsible for the biosynthesis of the {alpha}-1,6-glucan polymer. This {alpha}-1,6-glucosyltransferase protein is encoded by the HP0159 ORF. Both enzymes play an integral part in the biosynthesis of LPS, and insertional inactivation leads to the production of a truncated LPS molecule on the bacterial cell surface. The LPS structures were determined by mass spectrometry and chemical analyses. The linkage specificity of each glycosyltransferase was determined by nuclear magnetic resonance (NMR) analysis of model compounds synthesized in vitro. A cryogenic probe was used to structurally characterize nanomole amounts of the product of the HP1105 (JHP1032) enzyme. In contrast to the HP0159 enzyme, which displays the GT-8-predicted retaining stereochemistry for the reaction product, HP1105 (JHP1032) is the first member of this GT-8 family to have been shown to have an inverting stereochemistry in its reaction products.

Key words: CAZY 8 family / glycosyltransferases / Helicobacter pylori / lipopolysaccharide


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