Multi-wavelength anomalous dispersion data were collected to 2.7 Å (2.8 Å for the inflection wavelength) from a single crystal on station BM14 at the European Synchrotron Radiation Facility in Grenoble. The crystals were stabilized in 20% glycerol and 3 m sodium formate before flash cooling at 100 K. The crystals have six copies of the monomer in the asymmetric unit and a solvent content of 48%. The free enzyme crystallized in the space group F222 with cell dimensions a = 181.4 Å, b = 183.0 Å, c = 184.1 Å, and α = β = γ = 90°.
Crystals of native and selenomethionine-incorporated enzyme were obtained after two to five days of incubation at 17 ☌ using 2.7 to 3.0 m sodium formate as a precipitant. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid® resistance.Ĭrystallization and Data Collection-Crystallization trials were conducted using the hanging-drop method of vapor diffusion 5 μl of protein solution (0.15 to 0.25 m m in 1 m m Tris-HCl buffer, pH 7.5) was mixed with an equal volume of precipitant. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >10 5-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle.
Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 Å resolution in the absence of ligands, at 2.8 Å resolution bound to dalfopristin, and at 3.0 Å resolution in the presence of acetyl-coenzyme A. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination.
Synercid®, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. Glycobiology and Extracellular Matrices.
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