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因子的酶进行。这些酶中的一类， Fe-和2-酮戊二酸-（2 OG）依赖性酶，因其多功能性和催化 在相同的活性位点上发生不同的反应。该项目旨在研究一种较低的 Fe/2 OG依赖性酶的特征亚类-卤化酶。卤化酶，就像所有已知的 Fe/2 OG酶利用常见的氧化性铁基中间体[FeIV=O]2+，从 然后插入卤素离子或外源阴离子。卤素离子相对于 铁基中间体和底物还没有得到很好的理解;然而，这种取向已经涉及到铁基中间体和底物。 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 由稳定的钒氧离子[VIV O]2+模拟。当将其掺入羟化酶Fe/2 OG酶中时， 活性位点，它允许长时间的研究。在Fe/2 OG卤化酶中掺入氧钒提供了独特的 有机会研究基板的位置相对于金属和共基板参与 反应性这些研究将利用先进的光谱方法，同时整合非典型的 卤化酶活性位点内的氨基酸。与金属位点的非天然氨基酸配位可以提供 改变并潜在调整天然Fe/2 OG结合部分的反应性和产物形成的能力，同时 在钒基结合的络合物中引入独特的光谱比较。由此获得的知识 建议将导致对决定这些反应结果的因素的基本理解， 卤化酶活性位点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.