title: The molecular architecture of QdtA, a sugar 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum.
abstract: Unusual di- and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-D-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)-4-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP-sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3' hydroxyl and the C-4' keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP-sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, 53706.
authors.forename: James B
authors.initials: JB
authors.lastname: Thoden
authors.forename: Hazel M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D046950:Thermoanaerobacterium / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D003837:Deoxy Sugars; D002238:Carbohydrate Epimerases
keywords: 3,4-ketoisomerase; 3-acetamido-3,6-dideoxy-d-glucose; O-antigen; S-layer; cupin superfamily; dideoxysugar; enzyme mechanism; protein structure