Pfeifer Katarina, Van Cura Devon, Wu Kelvin J Y, Balskus Emily P
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
bioRxiv. 2025 May 28:2025.05.24.655970. doi: 10.1101/2025.05.24.655970.
Chemically reactive microbial natural products have enabled therapeutic development via their well-established bioactivities including anticancer, antibiotic, and antioxidant activities. However, discovery of reactive metabolites is particularly challenging because they may not tolerate traditional bioactivity-guided isolation workflows. Diazo-containing natural products are a subset of highly reactive microbial metabolites that display potent bioactivity and enable powerful (bio)synthetic transformations; however, instability of the diazo group to light, heat, mild acid, and mechanical shock has precluded their efficient discovery and application. Here, we develop a reactivity-based screening approach to capture diazo-containing metabolites and facilitate their discovery by mass spectrometry. This workflow revealed two novel diazo-containing natural products, 4-diazo-3-oxo-butanoic acid and diazoacetone, from the human lung pathogen . Biosynthetic investigations revealed a distinct enzymatic logic for diazo formation involving hydrazone oxidation catalyzed by the metalloenzyme Dob3, and biochemical characterization of Dob3 suggests promising future applications in biocatalysis. Overall, our work highlights the power of reactivity-guided strategies for identifying reactive metabolites and facilitating the discovery of unique enzymatic transformations.
具有化学反应性的微生物天然产物凭借其已确立的生物活性，包括抗癌、抗菌和抗氧化活性，推动了治疗药物的开发。然而，发现具有反应性的代谢产物极具挑战性，因为它们可能无法耐受传统的生物活性导向分离流程。含重氮的天然产物是一类具有高反应性的微生物代谢产物，它们表现出强大的生物活性，并能实现强大的（生物）合成转化；然而，重氮基团对光、热、弱酸和机械冲击的不稳定性阻碍了它们的有效发现和应用。在此，我们开发了一种基于反应性的筛选方法，以捕获含重氮的代谢产物，并通过质谱促进其发现。该工作流程从人类肺部病原体中揭示了两种新型含重氮天然产物，4-重氮-3-氧代丁酸和重氮丙酮。生物合成研究揭示了一种独特的重氮形成酶学逻辑，涉及金属酶Dob3催化的腙氧化，并且Dob3的生化特性表明其在生物催化方面具有广阔的应用前景。总体而言，我们的工作突出了反应性导向策略在识别反应性代谢产物和促进独特酶促转化发现方面的强大作用。
