The resistance of the emerging human pathogen Stenotrophomonas maltophilia to tetracycline antibiotics mainly depends on multidrug efflux pumps and ribosomal protection enzymes. However, the genomes of several strains of this Gram-negative bacterium code for a FAD-dependent monooxygenase (SmTetX) homologous to tetracycline destructases. This protein was recombinantly produced and its structure and function were investigated. Activity assays using SmTetX showed its ability to modify oxytetracycline with a catalytic rate comparable to those of other destructases. SmTetX shares its fold with the tetracycline destructase TetX from Bacteroides thetaiotaomicron; however, its active site possesses an aromatic region that is unique in this enzyme family. A docking study confirmed tetracycline and its analogues to be the preferred binders amongst various classes of antibiotics.

• Keywords
• FAD-dependent monooxygenases, antibiotic resistance, tetracycline,
• MeSH
• Anti-Bacterial Agents pharmacology   chemistry   MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Oxytetracycline * metabolism   MeSH
• Stenotrophomonas maltophilia * genetics   metabolism   MeSH
• Tetracycline pharmacology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Oxytetracycline * MeSH
• Tetracycline MeSH

Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396-His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp-His adduct mainly for binding and to a smaller extent for electron transfer.

• MeSH
• Bilirubin metabolism   MeSH
• Hypocreales metabolism   MeSH
• Protein Conformation MeSH
• Models, Molecular * MeSH
• Oxidation-Reduction MeSH
• Oxidoreductases Acting on CH-CH Group Donors metabolism   MeSH
• Electron Transport physiology   MeSH
• Protein Binding physiology   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• bilirubin oxidase MeSH Browser
• Bilirubin MeSH
• Oxidoreductases Acting on CH-CH Group Donors MeSH