PROJECT SUMMARY/ABSTRACT Tetracyclines are a vital class of antibiotics, but increasing resistance threatens their efficacy. Tetracycline re- sistance is thought to mainly occur through antibiotic efflux and ribosomal protection. Increasingly, resistance to late-generation tetracyclines, including antibiotics of last-resort, through enzymatic inactivation is being de- tected in environmental and clinical samples. These enzymes, known as tetracycline destructases, are now widely recognized as a clinically-relevant resistance mechanism. Despite recent interest in these enzymes, the precise sequence requirements that distinguish them from other flavoenzymes, and enable activity towards tetracyclines is unclear. To fill these gaps in knowledge, I will use a combination of bacterial genomics, syn- thetic biology, and molecular evolution. The long-term goal for this proposal is to better understand the evolu- tionary origins and structural features of tetracycline destructases in order to rationally design better diagnos- tics and inhibitors to re-store efficacy of this vital class of antibiotics before they become a widespread cause of morbidity and mortality. I propose two independent, yet complementary specific aims: (1) Characterize the se- quence-structure-function space of tetracycline destructases, and (2) Determine the capacity of related fla- voenzymes to evolve tetracycline inactivation activity. The first aim will test the hypothesis that the sequence determinants of FMO evolution toward tetracycline inactivation include regions that are structurally distal to substrate and cofactor binding sites. I will perform deep sequencing of randomized single-codon libraries of different tetracycline destructases that are selected for resistance to different generations of tetracycline antibi- otics and chemical inhibitors. Computational analysis of selected libraries will reveal sites on the enzymes that are heavily selected for substrate-specific activity. The second aim will test the hypothesis that tetracycline de- structases have evolutionary origins in tetracycline biosynthetic pathways. I will perform directed evolution on phylogenetically-related flavoenzymes using iterative cycles of mutagenesis and selection with tetracycline an- tibiotics until these enzymes confer levels of resistance that are comparable to tetracycline destructases. Se- quencing of clones will reveal the residues or domains which optimize these enzymes towards tetracycline ac- tivity. The proposed research is significant because antibiotic resistance is a public health crisis, and tetracy- cline destructases that degrade all known tetracyclines are already widely distributed in the environment. The proposed research is impactful because it provides a comprehensive and high-resolution understanding of the sequence-structure-function space of these enzymes, which will aid in the proactive development of co-thera- peutic and diagnostic agents that have the potential to mitiga...