Structural, mechanistic, & evolutionary characterization of tetracycline destructases

摘要 2015年，我们报道了四环素解构酶(TDase)的发现，这是一个能够 四环素(TET)抗生素的酶降解灭活，区别于经典的机械- Tet抗性的反常现象。自该报告以来，我们已在功能上将已知TDase池扩展到100 鉴定了酶，报道了许多TDase的晶体结构，并提出了一类小分子 对抗这些酶的抑制剂。TDase现在被广泛认为是一种与临床相关的耐药机制。 教条主义。这一提议的主要动机是更好地理解分子机制、进化 TDase的起源和结构特征，以便合理地设计更好的诊断和抑制剂来修复 随着TDase的继续传播并成为一种广泛的致病原因，一种至关重要的抗生素的有效性 发病率和死亡率。我们的合作努力已经产生了有影响力的科学成果，我们是理想的 有能力实现我们三个独立但互补的具体目标：1)阐明 TDase的Tet失活，2)了解TDase在遗传和种群水平上的进化，以及3) 开发TDase的抑制剂和诊断试剂。第一个目标将检验这样一种假设，即不同的亚 层结合方式和FAD辅因子取向决定了Tet氧化和调节的原子位置。 利用TDase的催化循环。我们认为我们可以将观察到的酶降解关联起来 利用X射线结晶学、光亲和交联法、酶动力学、 以及用各种Tdase和底物进行的同位素标记研究。第二个目标是检查序列 黄素单加氧酶进化为Tet失活的决定因素以及选择性Ad. TDase在表达不同机制的细菌群体中所提供的优势 测试阻力。我们将通过基于迭代序列的预测和表型来识别新的酶 验证，确定使用饱和突变和DNA改组进行活性所需的结构元件，以及 使用高通量的报告分析检查TDASS的人群水平的健康优势。第三 AIM将确定脱水四环素(ATC)类似物是否可以通过以下方式优化以抑制TDase 控制配基结合模式以及显色四联反应是否可作为诊断试剂 TDase在病原体中的表达。我们将使用我们开发的健壮的半合成方法进行修改 并用严格的生物化学分析、X-射线衍射仪研究得到的新型化合物。 结晶学和表型全细胞研究。这项拟议的研究意义重大，因为抗生素重新 耐药性是一种公共卫生危机，降解所有已知的四环素类药物的药物广泛分布于植物体内。 与环境和病原菌相反。拟议的研究具有影响力，因为它结合了Funda- 对协同治疗和诊断发展过程中酶的进化和机制的心智理解 有可能减轻酶促Tet失活所构成的新威胁的诊断试剂。

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 is impactful because it combines funda- mental understanding of enzyme evolution and mechanism with the development of co-therapeutic and diag- nostic agents that have the potential to mitigate the emerging threat posed by enzymatic Tet inactivation.