Understanding the Evolution of Halogenation in Biological Systems

了解生物系统中卤化的演变

• 批准号: 7792186
• 负责人: Emily Patricia Balskus
• 金额: $ 5.05万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7792186
• 关键词: Active Sites; Area; Asparagine; Biological; Biological Availability; Biological Factors; Cells; Chemicals; Chlorine; Development; Engineering; Enzymes; Evolution; Family; Halogens; Heme Iron; Hydrogen Bonding; Investigation; Libraries; Medicine; Metabolic; Methods; Mixed Function Oxygenases; Mutagenesis; Mutation; Nature; Oral; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Property; Research; Screening procedure; Techniques; Variant; biological systems; chlorination; computerized tools; directed evolution; ferryl iron; halogenation; interest; mutant; public health relevance; scaffold

DESCRIPTION (provided by applicant): Halogen atoms are important structural features found in a variety of natural and synthetic compounds of medicinal interest. Among enzymes used by biological systems to install halide functionality into natural products, the non-heme iron (II) halogenase family is of particular interest. These enzymes catalyze the chlorination of unactivated C-H bonds using a high valent iron (IV) oxo intermediate, a strategy shared by a related enzyme class, the non-heme iron (II) hydroxylases. The similar reactivity of these enzymes, as well as their structural homology, has led to the hypothesis that the halogenase family evolved from hydroxylases. As a means of probing the validity of this hypothesis, one can envision engineering a non-heme iron (II) hydroxylase into a halogenase. Two approaches for the engineering of the well-characterized asparagines hydroxylase AsnO are proposed: minimalist active site redesign and directed evolution. Minimalist active site redesign will employ structural, mechanistic and computational tools to identify selected target residues within AsnO for mutation. The directed evolution approach utilizes various mutagenesis techniques, including random and saturation mutagenesis, to generate a library of AsnO mutants, which will be screened for halogenase activity using one of two proposed spectrophotometric methods. The most promising variants will be subjected to further rounds of mutation and screening to provide an artificial halogenase suitable for structural and spectroscopic studies. PUBLIC HEALTH RELEVANCE Many of the halogenated metabolites found in nature possess intriguing biological activity which makes them interesting targets for the discovery and development of new medicines. In addition to naturally occurring compounds, many important man-made pharmaceuticals contain halides. In both cases, these atoms often have a dramatic, positive influence on the physiochemical properties of the molecule. Improvements in potency, oral bioavailability, cell-permeability, and metabolic stability may all result from the addition of a halide atom into a drug scaffold. As a result the development and mechanistic investigation of methods, both chemical and biological, for the selective installation of chlorine and other halogen atoms into molecules is an important area of research.

描述（由申请人提供）：卤素原子是在多种具有药用价值的天然和合成化合物中发现的重要结构特征。在生物系统用于将卤化物功能性安装到天然产物中的酶中，非血红素铁（II）卤化酶家族特别令人感兴趣。这些酶使用高价铁（IV）氧代中间体催化未活化的C-H键的氯化，这是相关酶类非血红素铁（II）羟化酶共享的策略。这些酶的相似反应性，以及它们的结构同源性，导致了卤化酶家族从羟化酶进化而来的假设。作为探测这一假设的有效性的手段，可以设想将非血红素铁（II）羟化酶工程化为卤化酶。提出了两种方法的工程的特点天冬酰胺羟化酶AsnO：最低限度的活性位点重新设计和定向进化。最低限度的活性位点重新设计将采用结构、机械和计算工具来鉴定AsnO内用于突变的选定靶残基。定向进化的方法利用各种诱变技术，包括随机和饱和诱变，产生一个库的AsnO突变体，这将被筛选的卤化酶活性使用两个建议的分光光度法之一。最有希望的变体将进行进一步的突变和筛选，以提供适合结构和光谱研究的人工卤化酶。公共卫生相关性自然界中发现的许多卤代代谢物具有有趣的生物活性，这使它们成为发现和开发新药的有趣目标。除了天然存在的化合物外，许多重要的人造药物都含有卤化物。在这两种情况下，这些原子通常对分子的物理化学性质产生显著的积极影响。效力、口服生物利用度、细胞渗透性和代谢稳定性的改善都可能是由于将卤素原子添加到药物支架中。因此，化学和生物学方法的发展和机理研究，用于选择性地将氯和其他卤素原子安装到分子中是一个重要的研究领域。