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J. Biol. Chem., Vol. 281, Issue 34, 24489-24495, August 25, 2006

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Structural Studies of FlaA1 from Helicobacter pylori Reveal the Mechanism for Inverting 4,6-Dehydratase Activity^*

Noboru Ishiyama¹, Carole Creuzenet², Wayne L. Miller^¶3, Melinda Demendi, Erin M. Anderson^¶, George Harauz^¶, Joseph S. Lam^¶4, and Albert M. Berghuis, Holds Canada Research Chairs in Structural Biology^||5

From the Department of Biochemistry and ^||Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, the Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, and the ^¶Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

FlaA1 from the human pathogen Helicobacter pylori is an enzyme involved in saccharide biosynthesis that has been shown to be essential for pathogenicity. Here we present five crystal structures of FlaA1 in the presence of substrate, inhibitors, and bound cofactor, with resolutions ranging from 2.8 to 1.9 Å. These structures reveal that the enzyme is a novel member of the short-chain dehydrogenase/reductase superfamily. Additional electron microscopy studies show the enzyme to possess a hexameric doughnut-shaped quaternary structure. NMR analyses of "real time" enzyme-substrate reactions indicate that FlaA1 is a UDP-GlcNAc-inverting 4,6-dehydratase, suggesting that the enzyme catalyzes the first step in the biosynthetic pathway of a pseudaminic acid derivative, which is implicated in protein glycosylation. Guided by evidence from site-directed mutagenesis and computational simulations, a three-step reaction mechanism is proposed that involves Lys-133 functioning as both a catalytic acid and base.

Received for publication, March 14, 2006 , and in revised form, April 27, 2006.

The atomic coordinates and structure factors (code) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). 2GN4, 2GN6, 2GN8, 2GN9, and 2GNA.

^* This work was supported in part by Collaborative Health Research Projects Grant 251007-02 from the Natural Sciences and Engineering Research Council of Canada (to J. S. L., C. C., and A. M. B.) and a Natural Sciences and Engineering Research Council of Canada operating grant (to G. H.). 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 Table 1 and supplemental Figs. 1 and 2.

¹ Recipient of a studentship from the Canadian Cystic Fibrosis Foundation.

² Recipient of a University Faculty Award from the Natural Sciences and Engineering Research Council of Canada and a Premier's Research Excellence Award from the Province of Ontario.

³ Recipient of a Doctoral Research Award from the Canadian Institute of Health Research.

⁴ Holds Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology.

⁵ To whom correspondence should be addressed: Depts. of Biochemistry and Microbiology and Immunology, McGill University, 740 Dr. Penfield Ave., Rm. 5202, Montreal, Quebec H3A 1A4, Canada. Tel.: 514-398-8795; Fax: 514-398-2036; E-mail: albert.berghuis{at}mcgill.ca.

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