Structural Analysis of QdtB, an Aminotransferase Required for the Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-α-d-glucose

摘要

3-Acetamido-3,6-dideoxy-R-D-glucose or Quip3NAc is an unusual deoxyamino sugar foundnin the O-antigens of some Gram-negative bacteria and in the S-layers of Gram-positive bacteria. It isnsynthesized in these organisms as a dTDP-linked sugar via the action of five enzymes. The focus of thisninvestigation is on QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, a PLP-dependentnaminotransferase that catalyzes the penultimate step in the production of dTDP-Quip3NAc. For this analysis,nthe enzyme was crystallized in the presence of its product, dTDP-Quip3N, and the structure was solvednand refined to 2.15 Å resolution. QdtB is a dimer, and its overall fold places it into the well-characterizednaspartate aminotransferase superfamily. Electron density corresponding to the bound product reveals thenpresence of a Schiff base between C-4′ of the PLP cofactor and the amino nitrogen of the sugar. Thosenamino acid side chains involved in binding the dTDP-sugar into the active site include Tyr 183, His 309,nand Tyr 310 from subunit 1 and Lys 219 from subunit 2. Notably there is a decided lack of interactionsnbetween the pyranosyl C-4′ hydroxyl of the dTDP-sugar and the protein. In keeping with this observation,nwe show that QdtB can also turn over dTDP-3-acetamido-3,6-dideoxy-R-D-galactose. This investigationnrepresents the first structural analysis of a sugar-modifying aminotransferase with a bound product in itsnactive site that functions at the C-3′ rather than the C-4′ position of the hexose.