Understanding the Evolution of Halogenation in Biological Systems

描述(由申请人提供)：卤素原子是在各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。在生物系统用来将卤化物功能安装到天然产品中的酶中，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键的氯化反应，这一策略被另一类相关的酶类--非血红素铁(II)羟基酶所共享。这些酶的相似反应活性以及它们的结构同源性导致了卤代酶家族是从羟基酶进化而来的假设。作为探索这一假说有效性的一种手段，人们可以设想将非血红素铁(II)羟基酶工程化为卤化酶。对天冬酰胺羟基酶ASNO的工程设计提出了两种方法：最低活性位重新设计和定向进化。最低限度的活性位点重新设计将使用结构、机械和计算工具来确定ASNO中选定的突变目标残基。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，来产生ASNO突变体文库，该文库将使用所提出的两种分光光度方法之一来筛选卤代酶活性。最有希望的变异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代酶。与公共健康有关自然界中发现的许多卤代谢物具有耐人寻味的生物活性，这使它们成为发现和开发新药的有趣目标。除了天然化合物外，许多重要的人造药物还含有卤化物。在这两种情况下，这些原子往往对分子的物理化学性质有戏剧性的积极影响。药效、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在药物支架中添加卤化物原子的结果。因此，化学和生物方法的发展和机理研究是将氯和其他卤素原子选择性安装到分子中的一个重要研究领域。

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.