Tetx2-A Tetracycline Inactivating Enzyme

Abstract

Resistance to the tetracycline antibiotics occurs primarily by efflux and ribosome protection mechanisms, however a tetracycline inactivating enzyme, TetX, was identified 15 years ago, although little is known about this mechanism. The gene encoding this enzyme was identified on a 𝘉𝘢𝘤𝘵𝘦𝘳𝘰𝘪𝘥𝘦𝘴 transposon and results from DNA sequence analysis and studies of bacterial culture media suggested that 𝘵𝘦𝘵𝘟's protein product might be a NADPH-requiring oxidase (𝘚𝘱𝘦𝘦𝘳 𝘉𝘚 𝘢𝘯𝘥 𝘚𝘢𝘭𝘺𝘦𝘳𝘴 𝘈𝘈. 𝘑 𝘉𝘢𝘤𝘵𝘦𝘳𝘪𝘢𝘭. 171(1): 148-153, 1989). We have expressed a copy of 𝘵𝘦𝘵𝘹 gene, 𝘵𝘦𝘵𝘹2, in Escherichia coli, and purified the enzyme to high purity. We showed that TetX2 is a monomeric 44 kDa cytoplasmic protein and UV-Vis and HPLC studies established that TetX contained an FAD cofactor. Continuous and stopped enzyme assays have been developed and established that that the enzyme requires 0₂ and NADPH for tetracycline degradation. Liquid chromatographic mass spectrometry (LC -MS) analysis of TetX reaction products using oxytetracycline (461 Da) as a substrate indicated that the enzyme catalyses the incorporation of one oxygen atom into oxytetracycline, resulting in a compound of 477 Da with no antibiotic activity. Steady state kinetic analysis demonstrated that TetX2 has a broad substrate specificity with the capacity to inactivate several members of the tetracycline family tested. Identification of the inactivated tetracycline product revealed that the tetracycline inactivation process is a TetX2 catalyzes tetracycline oxidation reaction. These studies provide the first biochemical analysis of a tetracycline inactivating enzyme.