Interrogating the Haloferryl State of Iron(II)- and 2-Oxoglutarate-Dependent Halogenases through Mimicry and Active Site Modifications

项目摘要 在自然界中，关键的细胞过程--例如转录、繁殖和小分子的产生 代谢物-由含有非血红素铁辅助因子的酶进行。这些酶中的一类是 铁和2-氧代磷酸(2OG)依赖的酶，以其多功能性和催化能力而闻名 同一活性部位内的不同反应。这个项目的目的是调查一种较少的 表征了依赖Fe/2OG的酶的亚类--卤代酶。卤素酶，就像我们所知道的 Fe/20OG酶，利用常见的氧化铁基中间体[FeIV=O]2，从 然后在衬底上插入卤化离子或外源阴离子。卤化物离子相对于原子的取向 铁基中间体和底物还不是很清楚；然而，这种取向已经被牵连在 产品的结果。最近的工作表明，不稳定的铁基中间体可以在结构上 由稳定的钒离子[VIV≡O]2模拟。当它结合到羟基酶Fe/2OG酶中时 活动站点，它允许长时间的学习。在Fe/20OG卤代酶中掺入钒提供了一种独特的 有机会调查衬底相对于金属和共衬底的位置 反应性。这些研究将利用先进的光谱方法，同时整合非正则的 卤酶活性部位内的氨基酸。非天然氨基酸对金属位置的配位可能提供 改变和潜在地调节天然Fe/20G结合部分中的反应性和产物形成的能力，而 引入了钒结合络合物中独特的光谱比较。通过这种方式获得的知识 提案将导致对决定反应结果的因素有一个基本的了解 卤代酶活性部位以及如何利用药物设计和合成的选择性反应性。

Project Summary Within Nature, key cellular processes – e.g. transcription, reproduction, and production of small molecule metabolites - are carried out by enzymes containing non-heme iron cofactors. One class of these enzymes, the Fe- and 2-oxogluterate- (2OG) dependent enzymes, are well-known for their versatility and ability to catalyze different reactions within the same active site. This project aims to investigate the reactivity of a less characterized subclass of Fe/2OG-dependent enzymes-the halogenases. The halogenases, like all known Fe/2OG enzymes, utilize the common oxidizing ferryl intermediate, [FeIV=O]2+, to abstract a hydrogen atom from the substrate and then insert a halide ion or exogenous anion. The orientation of the halide ion relative to the ferryl intermediate and substrate is not well understood; however, this orientation has been implicated in the outcome of the product. Recent work has indicated that the unstable ferryl intermediate can be structurally mimicked by the stable vanadyl ion, [VIV≡O]2+. When this is incorporated into a hydroxylase Fe/2OG enzyme active site, it allows for prolonged study. Incorporation of vanadyl in Fe/2OG halogenases offers a unique opportunity to investigate the position of the substrate in relation to the metal and the cosubstrates involved in reactivity. These studies will utilize advanced spectroscopic methods alongside integration of non-canonical amino acids within the halogenase active site. Unnatural amino acid coordination to the metal site may provide the ability to alter, and potentially tune, reactivity and product formation in the native Fe/2OG-bound moiety, while introducing unique spectroscopic comparisons in the vanadyl-bound complex. The knowledge gained by this proposal will lead to a fundamental understanding of factors that dictate the reaction outcome within these halogenase active sites and how to harness selective reactivity for drug design and synthesis.