Structural and Functional Studies of QdtC: An N-Acetyltransferase 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 dideoxy sugar found innthe O-antigens of various Gram-negative bacteria and in the S-layer glycoprotein glycans of some Grampositivenbacteria. It is produced in these organisms as a dTDP-linked sugar, with five enzymes ultimatelynrequired for its biosynthesis. The focus of this investigation is on the enzyme QdtC, a CoA-dependentnN-acetyltransferase that catalyzes the last step in the Quip3NAc biosynthetic pathway. For this analysis,nthree crystal structures were determined: the wild-type enzyme in the presence of acetyl-CoA and twonternary complexes of the enzyme with CoA and either dTDP-D-Quip3N or dTDP-3-amino-3,6-didexoy-nR-D-galactose (dTDP-D-Fucp3N). Each subunit of the trimeric enzyme is dominated by a left-handednu0001-helix motif with 11 turns. The three active sites are located at the subunit-subunit interfaces, and thentwo dTDP-sugar ligands employed in this study bind to the protein in nearly identical manners. Thosenresidues responsible for anchoring the hexose moieties of the dTDP-sugars to the protein include Glun141, Asn 159, and Asp 160 from one subunit and His 134 from another subunit. To probe the roles ofnvarious amino acid residues in the catalytic mechanism of the enzyme, 10 site-directed mutant proteinsnwere constructed and their kinetic parameters measured. On the basis of these data, a catalytic mechanismnis proposed for QdtC in which the acetylation of the sugar amino group does not require a catalytic basenprovided by the protein. Rather, the sulfur of CoA functions as the ultimate proton acceptor.