Heme-Dependent Chemistry in Tyrosine Oxidation

项目描述 血红素酶的一个显着特征是高价铁氧物种是介导 一系列显着的氧化反应。然而，一个难题是每种酶通常只促进一种 特定类型的反应。关键氧化剂形成后如何确定反应类型仍然是一个悬而未决的问题 这个问题的答案对我们对酶催化以及从头催化的基本理解有影响 酶设计和蛋白质工程。由于酪氨酸是天然产物的重要组成部分，因此 该应用重点关注三种血红素依赖性酪氨酸氧化酶的机制表征。每一个 这些酶利用单核血红素辅助因子氧化其基于酪氨酸的底物。有趣的是，一个 细胞色素 P450 蛋白（来自结核分枝杆菌的 CYP121）催化一种不寻常的氧化碳-碳 交叉偶联反应而不是更常见的羟基化反应。我们发现SfmD是该组织的新成员 色氨酸双加氧酶超家族，促进甲基化酪氨酸底物的区域选择性单加氧。 过氧化物酶 LmbB2 与组氨酸而不是半胱氨酸的轴向配体进行过氧化酶类型的反应。 这些酶不仅催化基于酪氨酸的氧化反应，而且还与抗菌药物有关 发展。鉴于血红素基氧化剂和底物结构的相似性，不可避免的 关于决定这些酶的催化活性的控制因素出现了问题。在目标#1中，我们 将决定 CYP121 的机械和结构特征。使用光谱和结构电池 方法与合成探针相结合，我们将揭示一种新颖的碳-碳偶联机制，该机制由 P450酶。在目标 2 中，我们将描述 SfmD 的结构和机制，重点是如何 底物定位于铁结合氧化剂并捕获催化中间体。我们已经确定了 该蛋白质是一种新型的基于血红素的加氧酶。目标#3 专注于研究催化的过氧化物酶反应 由 LmbB2 负责通过 L-酪氨酸形成 L-3,4-二羟基苯丙氨酸 (L-DOPA) 羟基化。我们将利用小分子探针来质疑机制假设。深入分析 这三个相关的催化系统将检验我们关于血红素结合氧化剂如何产生的假设，以及 针对芳香底物，揭示了看似的血红素酶的结构与功能关系 更高层次上不相关的超家族，并开发进一步帮助合理药物设计和开发潜在机制 发现过程。

PROJECT DESCRIPTION A distinguishing trait of heme enzymes is that a high-valent iron-oxo species is a common oxidant for mediating a remarkable array of oxidation reactions. However, one conundrum is that each enzyme in general promotes only a specific type of reaction. How the reaction type is determined after the formation of the key oxidant remains an open question whose answers have implications for our fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. Because tyrosine is an important building block of natural products, this application focuses on a mechanistic characterization of three heme-dependent tyrosine-oxidizing enzymes. Each of these enzymes employs a mononuclear heme cofactor to oxidize its tyrosine-based substrate. Intriguingly, a cytochrome P450 protein, CYP121 from Mycobacterium tuberculosis, catalyzes an unusual oxidative carbon-carbon cross-coupling reaction instead a more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes regioselective monooxygenation of a methylated tyrosine substrate. The peroxidase LmbB2 performs a peroxygenase-type of reaction with an axial ligand of histidine instead of cysteine. These enzymes not only catalyze tyrosine-based oxidation reactions but they are also related to antimicrobial drug development. Given the similarities of the heme-based oxidant and the structure of the substrates, the inevitable question arises regarding the governing factors that determine the catalytic activity of these enzymes. In Aim #1, we will determine the mechanistic and structural characteristics of CYP121. Using a battery of spectroscopic and structural approaches coupled with synthetic probes, we will unveil a novel carbon-carbon coupling mechanism mediated by the P450 enzyme. In Aim #2, we will characterize the structure and mechanism of SfmD with emphasis on how the substrate is positioned to the iron-bound oxidant and the capture of catalytic intermediates. We have already identified that this protein is a novel heme-based oxygenase. Aim #3 is focused on studying the peroxidase reaction catalyzed by LmbB2 that is responsible for L-3,4-dihydroxyphenylalanine (L-DOPA) formation through L-tyrosine hydroxylation. We will utilize small-molecule probes to interrogate mechanistic hypotheses. The in-depth analysis of these three related catalytic systems will test our hypothesis regarding how the heme-bound oxidant is generated and directed to the aromatic substrates, unravel the structure-function relationships of the heme enzymes of seemingly unrelated superfamilies at a higher level, and develop underlying mechanisms further aiding rational drug design and discovery processes.