Heme-Dependent Chemistry in Tyrosine Oxidation

项目描述 血红素酶的一个显著特征是高价铁氧物种是介导血红素合成的常见氧化剂。 一系列的氧化反应然而，一个难题是，每一种酶一般只促进一种蛋白质。 具体的反应类型。在关键氧化剂形成后，如何确定反应类型仍然是一个开放的问题。 这是一个问题，其答案对我们对酶催化的基本理解以及从头开始 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， 本申请集中于三种血红素依赖性酪氨酸氧化酶的机械表征。中的每 这些酶利用单核血红素辅因子来氧化其基于酪氨酸的底物。有趣的是，A 细胞色素P450蛋白，来自结核分枝杆菌的CYP 121，催化一种不寻常的氧化碳-碳 交叉偶联反应代替更常见的羟基化反应。我们发现SfmD是一个新的成员， 色氨酸双加氧酶超家族，其促进甲基化酪氨酸底物的区域选择性单加氧。 过氧化物酶LmbB 2与组氨酸而不是半胱氨酸的轴向配体进行过氧合酶型反应。 这些酶不仅催化基于酪氨酸的氧化反应，而且它们还与抗微生物药物相关。 发展考虑到血红素基氧化剂和底物结构的相似性， 关于决定这些酶的催化活性的支配因素产生了问题。在目标#1中，我们 将决定CYP 121的机制和结构特征。利用一组光谱和结构 结合合成探针的方法，我们将揭示一种新的碳-碳耦合机制， P450酶。在目标#2中，我们将重点描述SfmD的结构和机制， 基底定位于铁结合的氧化剂和催化中间体的捕获。我们已经确定 这种蛋白质是一种新的血红素加氧酶。目的#3专注于研究过氧化物酶催化的反应 LmbB 2通过L-酪氨酸负责L-3，4-二羟基苯丙氨酸（L-DOPA）的形成 羟基化我们将利用小分子探针来询问机制假说。深入分析 这三种相关的催化系统将检验我们关于血红素结合的氧化剂如何产生的假设， 针对芳香底物，解开血红素酶的结构-功能关系， 不相关的超家族在更高的水平，并开发潜在的机制，进一步帮助合理的药物设计， 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.