Understanding the Evolution of Halogenation in Biological Systems

了解生物系统中卤化的演变

• 批准号: 7483946
• 负责人: Emily Patricia Balskus
• 金额: $ 4.48万
• 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/7483946
• 关键词: Active Sites; Area; Asparagine; Biological; Biological Availability; Biological Factors; Cells; Chemicals; Chlorine; Class; Development; Engineering; Enzymes; Evolution; Family; Halogens; Heme Iron; Investigation; Libraries; Medicine; Metabolic; Methods; Mixed Function Oxygenases; Mutagenesis; Mutation; Nature; Object Attachment; Oral; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Property; Public Health; Research; Screening procedure; System; Techniques; Variant; chlorination; computerized tools; directed evolution; ferryl iron; halogenation; interest; mutant; 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）：卤素原子是在多种自然和合成化合物中发现的重要结构特征。在生物系统用来将卤化物功能安装到天然产物中的酶中，非血红素铁（II）卤素酶家族特别感兴趣。这些酶使用高价值铁（IV）氧中间体催化未激活的C-H键，这是相关酶类共享的策略，即非血红素铁（II）羟基酶。这些酶及其结构同源性的相似反应性导致了卤代酶家族从羟化酶演变而来的假设。作为探测该假设的有效性的一种手段，人们可以设想将非血红素铁（II）羟化酶工程化成卤化酶。提出了两种用于良好特征的天冬酰胺羟化酶ASNO的方法：简约的主动部位重新设计和定向进化。极简主义的主动站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。定向的进化方法利用各种诱变技术，包括随机和饱和诱变，生成一个ASNO突变体库，使用两种提议的分光光度法方法之一，将对卤素酶活性进行筛选。最有希望的变体将受到进一步的突变和筛查，以提供适合结构和光谱研究的人工卤化酶。公共卫生相关性的许多自然界中发现的卤代代谢产物具有有趣的生物学活性，这使它们成为发现和开发新药物的有趣目标。除了天然发生的化合物外，许多重要的人造药物还包含卤化物。在这两种情况下，这些原子通常对分子的生理化学特性都有巨大的积极影响。效力，口服生物利用度，细胞透露率和代谢稳定性的提高都可能是由于将卤化物原子添加到药物支架中而造成的。结果，对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学方法的开发和机理研究是重要的研究领域。