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

细菌位点特异性钒依赖性卤过氧化物酶的鉴定、表征和应用

• 批准号: 10663337
• 负责人: Shaun Mitchell Kirk McKinnie
• 金额: $ 37.28万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10663337
• 关键词: Biochemical; Biochemistry; Bioinformatics; Biological; Categories; Chemicals; Chemistry; Coenzymes; Ecology; Enzymatic Biochemistry; Enzymes; Family; Genome; Genomics; Halogens; Homologous Gene; Hydrogen Peroxide; Intention; Ions; Metabolism; Microbe; Modification; Natural Products; Nature; Organic Chemistry; Pathway interactions; Play; Production; Reaction; Reagent; Role; Site; Substrate Specificity; Techniques; Vanadates; Vanadium; aqueous; bioactive natural products; cofactor; halogenation; improved; microbial; novel; physical property; public repository; scaffold; secondary metabolite; small molecule; structural determinants; tool; wasting

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.

项目摘要 卤素原子（F-、Cl-、Br-、I-）在小有机分子中的掺入在以下方面起着重要作用： 调节它们的物理性质和生物活性，同时提供合成手柄， 化学修饰。在严格的反应条件下，卤素的区域选择性和对映选择性安装 化学合成方式在技术上具有挑战性，并且经常使用有毒试剂并产生 不期望的副产物。相比之下，自然已经开发了有效的酶策略， 卤素离子进入有机支架，废物产生可忽略不计。本提案侧重于探索 独特的卤化酶家族，特别是细菌位点特异性钒依赖性 卤代过氧化物酶（VHPOs），其使用配位的钒酸根离子（VO43-）和共底物过氧化氢， 氧化含水卤离子并将它们以区域和立体有择的方式安装在有机基底上。尽管 它们参与构建多种生物活性天然产物支架和催化化学多样性， 和有用的反应，没有额外的辅因子或辅酶，只有一小部分的数百个网站- 特异性VHPO同系物已被严格地表征。对这种定义不清的化学物质的探索 而生化空间是这个提议的智力驱动力。利用跨学科的化学，生物化学， 和基因组技术，我们的目标是更好地了解细菌位点特异性VHPO酶学通过三个 独立但相互关联的目标。第一个涉及新的基因组鉴定和分类 可在公共储存库中获得的VHPO同系物。改进了微生物位点的表示- 特异性卤化酶将使我们能够将基因组序列与最终的生物化学反应性联系起来， 直接从生物信息学特征预测化学的意图。第二个目标涉及 了解细菌次生代谢和化学生态学中未表征的VHPOs的作用。 大多数已知的细菌位点特异性同源物催化天然产物中的化学独特反应 生物合成途径;这些生物化学对于确定其同源产物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， 利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的次生代谢物的鉴定。 代谢物。最后，我们的目标是定义卤化物和有机底物特异性的结构决定因素， 合成应用，无论是在其天然底物或扩展到新的支架。这些目标将 同时提高我们对底物VHPO卤化酶学的理解， 大分子水平，并将促进生物催化的努力，应用这些位点特异性微生物卤化酶 化学上有用的转化。