title: Putative dioxygen-binding sites and recognition of tigecycline and minocycline in the tetracycline-degrading monooxygenase TetX.
abstract: Expression of the aromatic hydroxylase TetX under aerobic conditions confers bacterial resistance against tetracycline antibiotics. Hydroxylation inactivates and degrades tetracyclines, preventing inhibition of the prokaryotic ribosome. X-ray crystal structure analyses of TetX in complex with the second-generation and third-generation tetracyclines minocycline and tigecycline at 2.18 and 2.30 Å resolution, respectively, explain why both clinically potent antibiotics are suitable substrates. Both tetracyclines bind in a large tunnel-shaped active site in close contact to the cofactor FAD, pre-oriented for regioselective hydroxylation to 11a-hydroxytetracyclines. The characteristic bulky 9-tert-butylglycylamido substituent of tigecycline is solvent-exposed and does not interfere with TetX binding. In the TetX-minocycline complex a second binding site for a minocycline dimer is observed close to the active-site entrance. The pocket is formed by the crystal packing arrangement on the surface of two neighbouring TetX monomers. Crystal structure analysis at 2.73 Å resolution of xenon-pressurized TetX identified two adjacent Xe-binding sites. These putative dioxygen-binding cavities are located in the substrate-binding domain next to the active site. Molecular-dynamics simulations were performed in order to characterize dioxygen-diffusion pathways to FADH2 at the active site.
authors.affiliation: Department of Molecular Structural Biology, Institute for Biochemistry, University of Greifswald, Felix-Hausdorff-Strasse 4, Greifswald, Germany.
authors.forename: Gesa
authors.initials: G
authors.lastname: Volkers
authors.forename: João M
authors.initials: JM
authors.lastname: Damas
authors.forename: Gottfried J
authors.initials: GJ
authors.lastname: Palm
authors.forename: Santosh
authors.initials: S
authors.lastname: Panjikar
authors.forename: Cláudio M
authors.initials: CM
authors.lastname: Soares
authors.forename: Winfried
authors.initials: W
authors.lastname: Hinrichs
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000378:metabolism*; D001439:Bacteroides; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008911:Minocycline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000494:pharmacology*; D010100:Oxygen / Q000378:metabolism*; D013753:Tetracycline Resistance; D000078304:Tigecycline
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D000078304:Tigecycline; D006899:Mixed Function Oxygenases; D008911:Minocycline; D010100:Oxygen
keywords: TetX; antibiotic resistance; dioxygen diffusion; minocycline; molecular dynamics; monooxygenases; simulation; tigecycline; xenon