Structural analysis of QdtB, an aminotransferase required for the biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose.
3-Acetamido-3,6-dideoxy-alpha-d-glucose or Quip3NAc is an unusual deoxyamino sugar found in the O-antigens of some Gram-negative bacteria and in the S-layers of Gram-positive bacteria. It is synthesized in these organisms as a dTDP-linked sugar via the action of five enzymes. The focus of this investigation is on QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, a PLP-dependent aminotransferase that catalyzes the penultimate step in the production of dTDP-Quip3NAc. For this analysis, the enzyme was crystallized in the presence of its product, dTDP-Quip3N, and the structure was solved and refined to 2.15 A resolution. QdtB is a dimer, and its overall fold places it into the well-characterized aspartate aminotransferase superfamily. Electron density corresponding to the bound product reveals the presence of a Schiff base between C-4' of the PLP cofactor and the amino nitrogen of the sugar. Those amino acid side chains involved in binding the dTDP-sugar into the active site include Tyr 183, His 309, and Tyr 310 from subunit 1 and Lys 219 from subunit 2. Notably there is a decided lack of interactions between the pyranosyl C-4' hydroxyl of the dTDP-sugar and the protein. In keeping with this observation, we show that QdtB can also turn over dTDP-3-acetamido-3,6-dideoxy-alpha-d-galactose. This investigation represents the first structural analysis of a sugar-modifying aminotransferase with a bound product in its active site that functions at the C-3' rather than the C-4' position of the hexose.
Thoden
James B
JB
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Schäffer
Christina
C
Messner
Paul
P
Holden
Hazel M
HM
D003460:Crystallization; D018360:Crystallography, X-Ray; D003837:Deoxy Sugars / Q000096:biosynthesis*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011487:Protein Conformation; D012545:Schiff Bases; D021241:Spectrometry, Mass, Electrospray Ionization; D046950:Thermoanaerobacterium / Q000201:enzymology; D013942:Thymine Nucleotides / Q000096:biosynthesis*; D000637:Transaminases / Q000737:chemistry* / Q000378:metabolism
D003837:Deoxy Sugars; D012545:Schiff Bases; D013942:Thymine Nucleotides; C527679:dTDP-3-acetamido-3,6-dideoxyglucose; D000637:Transaminases