title: Structural and biochemical characterization of a bifunctional ketoisomerase/N-acetyltransferase from Shewanella denitrificans.
abstract: Unusual N-acetylated sugars have been observed on the O-antigens of some Gram-negative bacteria and on the S-layers of both Gram-positive and Gram-negative bacteria. One such sugar is 3-acetamido-3,6-dideoxy-α-d-galactose or Fuc3NAc. The pathway for its production requires five enzymes with the first step involving the attachment of dTMP to glucose-1-phosphate. Here, we report a structural and biochemical characterization of a bifunctional enzyme from Shewanella denitificans thought to be involved in the biosynthesis of dTDP-Fuc3NAc. On the basis of a bioinformatics analysis, the enzyme, hereafter referred to as FdtD, has been postulated to catalyze the third and fifth steps in the pathway, namely, a 3,4-keto isomerization and an N-acetyltransferase reaction. For the X-ray analysis reported here, the enzyme was crystallized in the presence of dTDP and CoA. The crystal structure shows that FdtD adopts a hexameric quaternary structure with 322 symmetry. Each subunit of the hexamer folds into two distinct domains connected by a flexible loop. The N-terminal domain adopts a left-handed β-helix motif and is responsible for the N-acetylation reaction. The C-terminal domain folds into an antiparallel flattened β-barrel that harbors the active site responsible for the isomerization reaction. Biochemical assays verify the two proposed catalytic activities of the enzyme and reveal that the 3,4-keto isomerization event leads to the inversion of configuration about the hexose C-4' carbon.
authors.affiliation: Department of Biochemistry, University of Wisconsin , Madison, Wisconsin 53706, United States.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Chantigian
authors.forename: James B
authors.initials: JB
authors.lastname: Thoden
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000096:biosynthesis*; D005643:Fucose / Q000031:analogs & derivatives* / Q000096:biosynthesis; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D064251:Multifunctional Enzymes / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D020592:Shewanella / Q000201:enzymology*; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: C478186:3-acetamido-3,6-dideoxy-D-galactose; D001426:Bacterial Proteins; D003837:Deoxy Sugars; D064251:Multifunctional Enzymes; D013942:Thymine Nucleotides; D005643:Fucose; D000123:Acetyltransferases; D002238:Carbohydrate Epimerases; D019746:Intramolecular Oxidoreductases; D000116:Acetylgalactosamine