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.

Markers Specific to Bacteroides fragilis Group Bacteria as Indicators of Anthropogenic Pollution of Surface Waters

“…In a study by Oravcova et al [ 75 ], enterococci harboring the van A gene were frequently identified in TWW, which indicates that these bacteria are not effectively eliminated during wastewater treatment. The high coefficients of correlation between the abundance of genes specific to BFG and genes encoding resistance to tetracyclines, MLS antibiotics, beta-lactams, and the bex A gene confirm previous findings that BFG bacteria are a major reservoir of these ARGs [ 24 , 76 , 77 ]. In the current study, close correlations were found between the prevalence of all analyzed ARGs in environmental samples (r = 0.63–0.98, p < 0.05).…”

Plasticity, dynamics, and inhibition of emerging tetracycline resistance enzymes

“…Surprisingly, chlortetracycline binds to Tet(50) in a ∼180° rotated orientation compared to the orientation in which chlortetracycline and other tetracycline substrates ( e.g. iodtetracycline, minocycline, tigecycline) bind Tet(X) 27,38 (Fig. 3a-d and Supplementary Fig.…”

Potential of Tetracycline Resistance Proteins To Evolve Tigecycline Resistance

“…TGC (orange) and FAD (gold) are indicated. The crystal structure of Tet(X) (PDB accession number 4A6N ) is based on data from reference 41 .…”

“…The other two amino acid residues were closer to the putative O 2 binding pockets. T280A has been previously suggested to interfere with O 2 diffusion ( 41 ). Therefore, other mutations (T281A, V285E, and L287V) from our screen that are located close to those hydrophobic pockets might also affect O 2 transport within the enzyme.…”