Evidence that SmTetX is not a tetracycline resistance determinant

SmTetX, a flavin-dependent monooxygenase from the opportunistic pathogen Stenotrophomonas maltophilia, is structurally related to the tetracycline-degrading Tet(X) resistance enzymes, though displays substantial divergence in active site residues. Here, we provide evidence that recombinant SmTetX is a functional monooxygenase, but the enzyme does not appear to recognize tetracycline antibiotics as substrates and does not catalyze their degradation. Furthermore, SmTetX fails to confer tetracycline resistance when heterologously expressed in E. coli. These findings indicate SmTetX is likely not a resistance determinant, presenting an opportunity to identify its physiological substrate and biological role.IMPORTANCEThe rapid dissemination of tet(X) resistance genes threatens the effectiveness of tetracycline antibiotics, including last-resort drugs such as tigecycline. Accurate assignment of functional Tet(X) homologs is therefore important for global antimicrobial resistance surveillance efforts and for selecting targets for the development of therapies that overcome Tet(X) resistance. Here, we demonstrate that SmTetX, a Tet(X)-like enzyme from Stenotrophomonas maltophilia, is likely not able to confer resistance to tetracyclines, indicating that its role in tetracycline resistance may have been misattributed. These findings highlight the need for robust experimental validation of novel tet(X) genes identified through sequence homology.