Understanding the Evolution of Halogenation in Biological Systems

描述（由申请人提供）：卤素原子是在各种天然和合成化合物中发现的重要结构特征，具有药用价值。在生物系统中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）氧中间体催化未活化的C-H键的氯化，这与相关酶类非血红素铁（II）羟化酶的策略相同。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。作为探索这一假设有效性的一种手段，人们可以设想将非血红素铁（II）羟化酶改造成卤化酶。本文提出了两种方法来设计具有良好特征的天冬酰胺羟化酶AsnO：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、机械和计算工具来确定AsnO中选择的目标残基进行突变。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，来生成AsnO突变体文库，这些突变体将使用两种提出的分光光度法中的一种来筛选卤素酶活性。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素酶。在自然界中发现的许多卤化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也含有卤化物。在这两种情况下，这些原子通常对分子的物理化学性质有显著的积极影响。药物的效力、口服生物利用度、细胞渗透性和代谢稳定性的改善都可以通过在药物支架中加入卤化物原子来实现。因此，将氯和其他卤素原子选择性地安装到分子中的化学和生物方法的发展和机理研究是一个重要的研究领域。

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.