# Structural, mechanistic, & evolutionary characterization of tetracycline destructases

> **NIH NIH U01** · WASHINGTON UNIVERSITY · 2024 · $681,709

## Abstract

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...

## Key facts

- **NIH application ID:** 10834137
- **Project number:** 5U01AI123394-09
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Gautam Dantas
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $681,709
- **Award type:** 5
- **Project period:** 2016-02-11 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10834137

## Citation

> US National Institutes of Health, RePORTER application 10834137, Structural, mechanistic, & evolutionary characterization of tetracycline destructases (5U01AI123394-09). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10834137. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*