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

通过模拟和活性位点修饰探究铁 (II) 和 2-氧化戊二酸依赖性卤化酶的卤铁基状态

• 批准号: 10226073
• 负责人: Jeffrey Worthington Slater
• 金额: $ 6.64万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10226073
• 关键词: Active Sites; Alanine; Alkylation; Amino Acids; Anabolism; Anions; Architecture; Azides; Bacteria; Behavior; Binding; Biochemical Reaction; Bromine; Cell physiology; Cells; Characteristics; Chlorine; Complex; Cyanides; DNA; Data; Deoxyribonucleotides; Development; Drug Design; Electron Spin Resonance Spectroscopy; Engineering; Enzymes; Frequencies; Genetic Transcription; Halogens; Health; Histidine; Human; Hydrogen; Hydrogen Bonding; Hydroxylation; Individual; Ions; Iron; Isotopes; Kinetics; Knowledge; Life; Ligands; Location; Metalloproteins; Metals; Methods; Mixed Function Oxygenases; Modification; Natural Product Drug; Nature; Outcome; Oxides; Oxygen; Oxygenases; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Physiologic pulse; Plants; Positioning Attribute; Process; Production; Proteins; RNA; Reaction; Regulation; Reporting; Reproduction; Ribonucleotides; Site; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Structure; Succinates; Techniques; Time; Vanadyl; Variant; Work; X-Ray Crystallography; alpha ketoglutarate; analog; carboxylate; chemical synthesis; cofactor; drug synthesis; epimerization; functional group; fungus; halogenation; member; mimicry; novel; pathogenic microbe; prevent; repaired; small molecule; unnatural amino acids; vibration

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.

项目概要 在自然界中，关键的细胞过程——例如小分子的转录、复制和生产 代谢物 - 由含有非血红素铁辅助因子的酶进行，其中一类是酶。 Fe- 和 2-酮戊二酸 (2OG) 依赖性酶以其多功能性和催化能力而闻名 同一活性位点内的不同反应该项目旨在研究较少的反应性。 与所有已知的一样，Fe/2OG依赖性酶的特征亚类——卤化酶。 Fe/2OG 酶，利用常见的氧化铁中间体 [FeIV=O]2+，从 基板，然后插入卤化物离子或外源阴离子 卤化物离子相对于的方向。 Ferrel 中间体和底物还没有被很好地理解；然而，这种方向已经与 最近的工作表明不稳定的费雷尔中间体可以在结构上发生变化。 当将其掺入羟化酶 Fe/2OG 时，可通过稳定的氧钒基离子 [VIVeqO]2+ 进行模拟。 活性位点，它允许在 Fe/2OG 卤化酶中进行长时间的研究。 有机会研究基材相对于金属和涉及的共基材的位置 这些研究将利用先进的光谱方法以及非规范的整合。 卤化酶活性位点内的氨基酸可以提供与金属位点的非天然氨基酸配位。 改变并可能调整天然 Fe/2OG 结合部分的反应性和产物形成的能力，同时 在氧钒结合络合物中引入独特的光谱比较由此获得的知识。 提案将导致对决定这些反应结果的因素有一个基本的了解 卤化酶活性位点以及如何利用选择性反应性进行药物设计和合成。