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) 氧化中间体催化未激活的 C-H 键的氯化，这是相关酶类非血红素铁 (II) 羟化酶所共有的策略。这些酶的相似反应性及其结构同源性导致了卤化酶家族从羟化酶进化而来的假设。作为探讨这一假设有效性的一种方法，人们可以设想将非血红素铁 (II) 羟化酶改造为卤化酶。提出了两种设计充分表征的天冬酰胺羟化酶 AsnO 的方法：极简活性位点重新设计和定向进化。极简活性位点重新设计将采用结构、机械和计算工具来识别 AsnO 中选定的目标残基以进行突变。定向进化方法利用各种诱变技术，包括随机诱变和饱和诱变，生成 AsnO 突变体文库，将使用两种提议的分光光度方法之一筛选卤化酶活性。最有希望的变体将接受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。公众健康相关性 自然界中发现的许多卤化代谢物具有令人着迷的生物活性，这使得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也含有卤化物。在这两种情况下，这些原子通常会对分子的理化性质产生巨大的积极影响。在药物支架中添加卤化物原子可能会提高药效、口服生物利用度、细胞渗透性和代谢稳定性。因此，用于将氯和其他卤素原子选择性安装到分子中的化学和生物方法的开发和机理研究是一个重要的研究领域。