Crystal structure of TDP-fucosamine acetyltransferase (WecD) from Escherichia coli, an enzyme required for enterobacterial common antigen synthesis.
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)-alpha-d-Fuc4NAc-(1-->4)-beta-d-ManNAcA-(1-->4)-alpha-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-Angstrom resolution and bound to acetyl-CoA at a 1.66-Angstrom 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.
Hung
Ming-Ni
MN
Biotechnology Research Institute, 6100 Royalmount Ave., Montreal QC, Canada H4P 2R2.
Rangarajan
Erumbi
E
Munger
Christine
C
Nadeau
Guy
G
Sulea
Traian
T
Matte
Allan
A
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis; D001665:Binding Sites; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D000942:Antigens, Bacterial; D029968:Escherichia coli Proteins; C017286:enterobacterial common antigen; D000105:Acetyl Coenzyme A; D005647:Fucosyltransferases