Exploring and Controlling Phenazine Biosynthesis with Chemical Biology

非那嗪是一类具有氧化还原活性的细菌次生代谢物，作为抗生素和毒力因子对细胞存活起重要作用。因此，它们的生物合成可能是治疗传染病的新药的一个有趣的靶点。非那嗪类药物通过一个保守的途径由chorismic酸产生，该途径涉及到phzoperon编码的酶。尽管对这一过程的总体理解有所进展，但重要的细节仍不清楚，因为非那嗪的生物合成是通过几种难以表征的不稳定中间体进行的。本提案旨在通过将合成化学与生化和结构技术相结合来填补现有的空白。特别是，申请人建议(1)研究2-氨基-2-去氧异氯酸酯（ADIC）合成酶PhzE与相关的邻氨基甲酸酯合成酶之间的差异，后者将ADIC进一步转化为邻氨基甲酸酯，尽管它们具有非常相似的活性中心；(2)深入了解PhzF催化的[1,5]-原向性转移可能遵循周环反应机制；(3)将现有的PhzB类似物开发成非那嗪生物合成抑制剂；(4)了解PhzA/PhzB异源二聚化在假单胞非那嗪生产商中的生化相关性。Blankenfeldt博士（德国多特蒙德MPI）和Breinbauer博士教授（奥地利格拉茨理工大学）的合作确保在三年资助期内实现这些目标。合作伙伴在有机合成、生物化学和结构生物学方面拥有互补的专业知识，近年来，他们通过在非那嗪生物合成领域做出重大贡献，展示了富有成效的合作。跨国资助将使两名博士生能够在方法学上不同的领域获得广泛的专业知识，拟议的研究将作出重大贡献，不仅提供新的酶机制的详细描述，而且还提供可以用作铅结构的工具化合物，以对抗由产非那嗪的微生物造成的传染病。

The phenazines are a large class of redox-active bacterial secondary metabolites that play an important role for cell survival by acting as antibiotics and virulence factors. Their biosynthesis may therefore be an interesting target for novel pharmaceuticals in the treatment of infectious disease. Phenazines derive from chorismic acid through a conserved pathway that involves enzymes encoded in the phzoperon. Despite progress in the general understanding of the process, important details are still unclear due to the fact that phenazine biosynthesis proceeds through several unstable intermediates that are difficult to characterize. This proposal aims at filling the existing gaps by combining synthetic chemistry with biochemical and structural techniques.In particular, the applicants propose to(1) investigate differences between 2-amino-2-desoxyisochorismate (ADIC) synthase PhzE and the related anthranilate synthases, which convert ADIC further to anthranilate despite having a very similar active center;(2) gain detailed insight into PhzF, which catalyzes a [1,5]-prototropic shift that may follow a pericyclic reaction mechanism;(3) develop existing product analogues of PhzB into inhibitors of phenazine biosynthesis and(4) understand the biochemical relevance of PhzA/PhzB heterodimerization in pseudomonal phenazine producers.The collaboration of Dr. Blankenfeldt (MPI Dortmund, Germany) and Prof. Dr. Breinbauer (Graz University of Technology, Austria) ensures that these aims will be met within the three years funding period. The partners possess complimentary expertises in organic synthesis, biochemistry and structural biology, and they have demonstrated a productive collaboration by contributing significantly to the field of phenazine biosynthesis in recent years. Transnational funding will enable two Ph.D. students to acquire a broad expertise in methodologically heterogeneous areas, and the proposed research will make significant contributions not only by providing detailed descriptions of novel enzyme mechanisms but also tool compounds that can be employed as lead structures against infectious disease inflicted by phenazine-producing microorganisms.