Biosynthesis of unusual bio-orthogonal functionalities in natural products

天然产物是研究、治疗甚至引起人类疾病的重要小分子，它们通常具有对其生物活性至关重要的独特官能团。通过利用合成这些功能的生物合成机制，可以增强、改变或减少母体化合物的生物活性，并将生物合成机制应用于新的系统中，以安装官能团。为了实现这一目标，天然产物生物合成的化学逻辑和酶机制需要得到充分的表征和理解。本项目将重点研究天然产物中不寻常的药团（包括末端炔、异腈和n -羟基三氮）的生物合成酶的功能表征和机械分析。独特的金属酶已被确定为重要的生物合成酶，包括膜结合的二铁依赖的双功能去饱和酶/乙酰化酶生成炔，非血红素铁和α-酮戊二酸依赖的氧化酶/脱羧酶异腈合成，和双结构域酶组成的c端甲硫基trna合成酶样结构域和n端cupin结构域形成n-羟基三氮烯。对这些新发现的酶的生物化学和机制了解仍然很差，需要在本项目中进行深入研究。除了作为生物活性天然产物的弹头外，这些功能具有独特的化学和物理性质，并用于各种化学生物学应用的生物正交化学转化。因此，我们将进一步探索他们的生物合成机器的应用，根据需要在各种生物分子上安装这些“可点击”的功能。研究策略在选择新颖功能及其独特但鲜为人知的生物合成机制、多学科研究工具和直接针对健康相关应用的目标导向方法方面具有创新性。特别是，我们在方法设计中超越了酶和途径表征，追求这些独特酶在灵活性方面的应用，以对人类福祉产生积极影响。因此，由于对天然产物酶学领域的影响以及在基础和转化层面的生物医学和生物技术研究中的应用，拟议的研究具有重要意义。

Natural products are important small molecules for studying, treating, and even causing human diseases, and they typically have unique functional groups that are critical for their biological activities. By exploiting the biosynthetic machinery by which these functionalities are synthesized, it is possible to enhance, vary or diminish the biological activities of parent compounds and apply the biosynthetic machinery to new systems for functional group installation. Toward this goal, the chemical logic and enzymatic machinery underlying natural product biosynthesis need to be fully characterized and understood. This project will focus on functional characterization and mechanistical interrogation of enzymes responsible for biosynthesis of unusual pharmacophores of natural products, including terminal alkyne, isonitrile and N-hydroxytriazene. Unique metalloenzymes have been identified important for their biosynthesis, including the membrane-bound di-iron dependent bifunctional desaturase/acetylenase for alkyne generation, the non-heme iron and α-ketoglutarate dependent oxidase/decarboxylase for isonitrile synthesis, and a didomain enzyme consisting of a C-terminal methionyl-tRNA synthetase-like domain and an N-terminal cupin domain for N-hydroxytriazene formation. The biochemical and mechanistic understanding of these newly discovered enzymes remain poor, warranting an in-depth study in this project. In addition to serving as the warheads of bioactive natural products, these functionalities have distinct chemical and physical properties and are utilized in bio-orthogonal chemical transformations for various chemical biology applications. We will thus further explore the application of their biosynthetic machinery to install these “clickable” functionalities on various biomolecules on demand. The research strategy is innovative in terms of the choice of novel functionalities and their unique but poorly understood biosynthetic machinery, the multidisciplinary research tools, and the goal-oriented approaches to directly target health-related applications. In particular, we go beyond the enzyme and pathway characterization in the approach design, pursuing applications of these unique enzymes in terms of flexibility to have a positive impact on human well-being. The proposed research is thus significant due to both implications for the field of natural product enzymology and utilities in biomedical and biotechnological research at the basic and translational levels.