ABSTRACT In 2015, we reported the discovery of the tetracycline destructases (TDases), a family of flavoenzymes capable of inactivating tetracycline (Tet) antibiotics by enzymatic degradation, distinguishing them from canonical mech- anisms of Tet resistance. Since that report we have expanded the pool of known TDases to >100 functionally identified enzymes, reported crystal structures of numerous TDases, and proposed a class of small molecule inhibitors to combat these enzymes. TDases are now widely recognized as a clinically-relevant resistance mech- anism. The central motivation for this proposal is to better understand the molecular mechanisms, evolutionary origins, and structural features of TDases in order to rationally design better diagnostics and inhibitors to restore efficacy of a vital class of antibiotics as TDases continue to disseminate and become a widespread cause of morbidity and mortality. Our collaborative effort has yielded impactful scientific results, and we are ideally equipped to carry out our three independent yet complementary specific aims: 1) Elucidate the mechanism of Tet inactivation by the TDases, 2) Understand the evolution of TDases at genetic and population levels, and 3) Develop inhibitors and diagnostic agents for TDases. The first aim will test the hypothesis that diverse sub- strate-binding modes and FAD cofactor orientations determine the atomic site of Tet oxidation and reg- ulate the catalytic cycle of the TDases. We propose that we can correlate observed enzymatic degradation products with respective binding modes using X-ray crystallography, photoaffinity crosslinking, enzyme kinetics, and isotopic labeling studies with a variety of TDases and substrates. The second aim examines the sequence determinants of flavin monooxygenase evolution toward Tet inactivation as well as the selective ad- vantage that TDases provide in the context of bacterial populations expressing different mechanisms of Tet resistance. We will identify novel enzymes through iterative sequence-based predictions and phenotypic validation, identify structural elements required for activity using saturation mutagenesis and DNA shuffling, and examine the population-level fitness advantages of TDases using high-throughput reporter assays. The third aim will determine whether anhydrotetracycline (aTC) analogs can be optimized to inhibit TDases by controlling ligand binding mode and whether chromogenic Tets can serve as diagnostic agents for TDase expression in pathogens. We will use robust semi-synthetic methods developed by us for modification of the Tet and aTC scaffolds and study the resulting novel compounds with rigorous biochemical assays, X-ray crystallography, and phenotypic whole-cell studies. The proposed research is significant because antibiotic re- sistance is a public health crisis, and TDases that degrade all known tetracyclines are widely distributed in di- verse environmental and pathogenic bacteria. The proposed research i...