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J. Biol. Chem., Vol. 281, Issue 13, 8907-8916, March 31, 2006

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Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter pylori^*

Ian C. Schoenhofen, Vladimir V. Lunin, Jean-Philippe Julien^¶, Yunge Li, Eunice Ajamian^¶, Allan Matte, Miroslaw Cygler^¶, Jean-Robert Brisson, Annie Aubry, Susan M. Logan, Smita Bhatia, Warren W. Wakarchuk, and N. Martin Young¹

From the Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, the Biotechnology Research Institute, National Research Council, Montreal, Quebec H4P 2R2, and the ^¶Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP--D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy--L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy--L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy--L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a ⁴C₁ chair conformation, which is different from the predominant ¹C₄ form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the -L-arabino intermediate during the amino transfer reaction. The essential role of Lys¹⁸³ in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

Received for publication, December 5, 2005 , and in revised form, January 12, 2006.

The atomic coordinates and structure factors (codes 2FN6, 2FNI, and 2FNU) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the National Research Council's Genomics and Health Initiative and by a grant from the Canadian Institutes of Health Research (Grant 200103GSP-90094-GMX-CFAA-19924 to M. C.). 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.

¹ To whom correspondence should be addressed: Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada. Tel.: 613-990-0855; Fax: 613-941-1327; E-mail: Martin.Young{at}nrc-cnrc.gc.ca.

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