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J. Biol. Chem., Vol. 275, Issue 45, 34873-34880, November 10, 2000
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From the FlaA1 is a small soluble protein of unknown
function in Helicobacter pylori. It has homologues that are
essential for the virulence of numerous medically relevant bacteria.
FlaA1 was overexpressed as a histidine-tagged protein and purified to
homogeneity by nickel chelation and cation exchange chromatography.
Spectrophotometric assays, capillary electrophoresis, and mass
spectrometry analyses showed that FlaA1 is a novel bifunctional
C6 dehydratase/C4 reductase specific for
UDP-GlcNAc. It converts UDP-GlcNAc into a
UDP-4-keto-6-methyl-GlcNAc intermediate, which is stereospecifically
reduced into UDP-QuiNAc. Substrate conversions as high as 80%
were obtained at equilibrium. The Km and
Vmax for UDP-GlcNAc were 159 µM
and 65 pmol/min, respectively. No exogenous cofactor was required to
obtain full activity of FlaA1. Additional NADH was only used with poor
efficiency for the reduction step. The biochemical characterization of
FlaA1 is important for the elucidation of biosynthetic pathways that lead to the formation of 2,6-deoxysugars in medically relevant bacteria. It establishes unambiguously the first step of the pathway and provides the means of preparing the substrate UDP-QuiNAc, which is
necessary for the study of downstream enzymes.
FlaA1, a New Bifunctional UDP-GlcNAc C6
Dehydratase/ C4 Reductase from Helicobacter
pylori*
§,
University of Guelph, Department of
Microbiology, Guelph, Ontario N1G 2W1, Canada and the ¶ National
Research Council, Ottawa, Ontario K1A 0R6, Canada
*
This work was supported in part by Medical Research Council
of Canada Grant MT14687 (to J. S. L.) and the Canadian Bacterial Diseases Network (a consortium of the Federal Networks of Centers of
Excellence).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 519 824 4120 (ext. 3823); Fax: 519 837 1802; E-mail: jlam@uoguelph.ca.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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