Matthew J. Beech

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. IMPORTANCE: The 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.

Use of the clinically vital β-lactam antibiotics is increasingly compromised by resistance, commonly mediated by β-lactamases. While clinically used serine-β-lactamase (SBL) inhibitors have long been available, metallo-β-lactamase (MBL) inhibitors are not yet approved for clinical use. We report the structure-guided development of pyrrole-2-carboxylic acid derivatives as potent inhibitors of the clinically important di-Zn(II) ion containing B1 MBLs (NDM-1, VIM-1, VIM-2, IMP-1). Crystallographic studies reveal the pyrrole-2-carboxylic acids inhibit B1 MBLs via active site Zn(II)-coordination of the inhibitor carboxylate and trapping of the di-Zn(II) ion bridging hydroxide, the latter of which reacts with the substrate β-lactam ring during hydrolysis. Appropriately derivatized pyrrole-2-carboxylic acids enhance the activity of carbapenems against MBL producing Gram-negative clinical isolates. The results support further development of metalloenzyme inhibitors that exploit binding to structural or catalytically important water molecules, an approach which may help in achieving selectivity over other metalloenzymes compared to metal-chelation based approaches.

The Tet(X) flavin-dependent monooxygenases enable tetracycline antibiotic resistance by catalysing inactivating hydroxylation, so preventing inhibition of bacterial ribosomes. Tet(X) resistance is growing rapidly, threatening the efficacy of important last-resort tetracyclines such as tigecycline. Tet(X) inhibitors have potential to protect tetracyclines in combination therapies, but their discovery has been hampered by lack of high-throughput assays. We report the development of an efficient fluorescence polarisation Tet(X) binding assay employing a tetramethylrhodamine-glycyl-minocycline conjugate that enables inhibitor discovery. The assay was applied to tetracycline substrates and reported inhibitors, providing insight into their binding modes. Screening of a bioactive molecule library identified novel Tet(X) inhibitors, including psychoactive phenothiazine derivatives and the 5-HT₄ agonist tegaserod, the activities of which were validated by turnover assays. Crystallographic studies of Tet(X4)-inhibitor complexes reveal two new inhibitor binding modes, importantly providing evidence for active site binding of Tet(X) inhibitors that do not share structural similarity with tetracycline substrates. In some cases, potentiation of tigecycline activity was observed in bacteria expressing Tet(X4). The combined results provide non-tetracycline scaffolds for development of potent Tet(X) inhibitors and highlight the need to evaluate the impact of non-antibiotics on antimicrobial resistance.

The cephalosporin antibiotics cefiderocol, ceftazidime and cefepime undergo fragmentation on reaction with penicillin binding protein 3 from Pseudomonas aeruginosa.

Polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) multienzymes produce numerous high value metabolites. The protein subunits which constitute these megasynth(et)ases must undergo ordered self-assembly to ensure correct organisation of catalytic domains for the biosynthesis of a given natural product. Short amino acid regions at the N- and C-termini of each subunit, termed docking domains (DDs), often occur in complementary pairs, which interact to facilitate substrate transfer and maintain pathway fidelity. This review details all structurally characterised examples of NRPS and PKS DDs to date and summarises efforts to utilise DDs for the engineering of biosynthetic pathways.