Identification, characterization, and application of bacterial site-specific vanadium-dependent haloperoxidase enzymes

项目摘要 在有机小分子中，卤素原子(F-、Cl-、Br-、I-)的掺入在 调节它们的物理性质和生物活性，同时为其他 化学修饰。严格意义上的卤素的区域选择性和对映选择性安装 化学合成方法在技术上具有挑战性，经常使用有毒试剂并产生 不良副产品。相比之下，大自然已经开发出有效的酶策略来结合水 卤素离子进入有机支架，产生的废物可以忽略不计。这项建议集中在探索一种 独特的卤化酶家族，特别是依赖于细菌部位的钒 卤代过氧酶(VHPO)，它使用一个配位的钒离子(VO43-)和共底物过氧化氢来 氧化含水的卤化物离子，并以区域和立体特定的方式将它们安装在有机基质上。尽管 它们参与构建多种生物活性天然产物支架并催化不同的化学成分 和有用的反应，没有额外的辅因子或辅酶，只有数百个位点中的一小部分- 对特定的VHPO同系物进行了严格的表征。对这种定义模糊的化学物质的探索 生化空间是推动这一提议的智力因素。使用跨学科的化学、生物化学、 和基因组技术，我们的目标是通过三种方法更好地了解细菌位点特异性的VHPO酶 独立但又相互关联的目标。第一部分涉及小说的基因组鉴定和分类 VHPO同系物可在公开可用的存储库中获得。改善了微生物位置的表现- 特定的卤代酶将允许我们将基因组序列与生化反应联系起来，最终 直接从生物信息学信号预测化学成分的意图。第二个目标涉及 了解未特化的VHPO在细菌次级代谢和化学生态学中的作用。 已知的大多数细菌定点同系物在天然产物中催化独特的化学反应。 生物合成途径；这些生物化学对于确定其同源产品的生物活性至关重要。 我们认为，新的VHPO反应性和不同的底物支架仍有待发现，并且 使用同源基因作为生物合成的‘钩子’将有助于基于基因组的新次级细胞的鉴定 代谢物。最后，我们的目标是定义卤化物和有机底物专一性的结构决定因素 在天然基质中或扩展到新型支架中的合成应用。这些目标将 同时提高我们对VHPO底物卤化酶学的认识 大分子水平，并将促进生物催化努力，以应用这些特定部位的微生物卤代酶 朝着化学上有用的转变。

Project Summary The incorporation of halogen atoms (F-, Cl-, Br-, I-) in small organic molecules plays a significant role in modulating their physical properties and biological activities while providing a synthetic handle for additional chemical modification. The regioselective and enantioselective installation of halogens in a strictly chemosynthetic manner is technically challenging and frequently utilizes toxic reagents and generates undesirable byproducts. In contrast, Nature has developed efficient enzymatic strategies to incorporate aqueous halide ions into organic scaffolds with negligible waste production. This proposal focuses on the exploration of a unique family of halogenating enzymes, specifically the bacterial site-specific vanadium dependent haloperoxidases (VHPOs), that use a coordinated vanadate ion (VO43-) and co-substrate hydrogen peroxide to oxidize aqueous halide ions and install them in a regio- and stereospecific manner on organic substrates. Despite their involvement in constructing multiple bioactive natural product scaffolds and catalyzing chemically diverse and useful reactions without additional cofactors or coenzymes, only a small fraction of the hundreds of site- specific VHPO homologs have been rigorously characterized. The exploration of this poorly defined chemical and biochemical space is what intellectually drives this proposal. Using interdisciplinary chemical, biochemical, and genomic techniques, we aim to better understand bacterial site-specific VHPO enzymology through three independent, yet interrelated objectives. The first involves the genomic identification and categorization of novel VHPO homologs available within publicly available repositories. Improved representation of microbial site- specific halogenases will permit us to correlate genomic sequences to biochemical reactivities with the ultimate intention of predicting chemistries directly from bioinformatic signatures. The second objective involves understanding the roles of uncharacterized VHPOs within bacterial secondary metabolism and chemical ecology. The majority of known bacterial site-specific homologs catalyze chemically unique reactions in natural product biosynthetic pathways; these biochemistries are critical for establishing the bioactivities of their cognate products. We propose that novel VHPO reactivities and diverse substrate scaffolds remain to be discovered, and that the use of homologous genes as biosynthetic ‘hooks’ will facilitate the genome-based identification of new secondary metabolites. Finally, we aim to define the structural determinants of halide and organic substrate specificity for synthetic applications, either within their native substrates or expanded to novel scaffolds. These objectives will simultaneously improve our understanding of VHPO halogenation enzymology at the substrate and macromolecular level and will facilitate biocatalytic efforts to apply these site-specific microbial halogenases towards chemically useful transformations.