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Journal of Bacteriology, August 2006, p. 5606-5617, Vol. 188, No. 15
0021-9193/06/$08.00+0     doi:10.1128/JB.00306-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Crystal Structure of TDP-Fucosamine Acetyltransferase (WecD) from Escherichia coli, an Enzyme Required for Enterobacterial Common Antigen Synthesis

Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada,¹ Department of Biochemistry, McGill University, Montreal, Quebec Canada²

Received 1 March 2006/ Accepted 22 May 2006

Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-D-glucosamine, N-acetyl-D-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-D-galactose, organized into trisaccharide repeating units having the sequence 3)--D-Fuc4NAc-(14)-ß-D-ManNAcA-(14)--D-GlcNAc-(1. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-D-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.