Heme-dependent chemistry in tyrosine oxidation: Diversity Supplement on 5R01GM108988-08 for supporting Samuel Montoya

母研究项目摘要 血红素铁依赖酶的标志之一是它可以进行广泛的氧化反应， 用高价铁氧物种催化。然而，一个难题是，一般来说， 只会引起某种特定的反应。反应类型是如何确定后形成的 临界氧化剂仍然是一个悬而未决的问题，其答案对基本的 了解酶催化以及从头酶设计和蛋白质工程。因为 酪氨酸是天然产物的重要组成部分，该项目侧重于一种机制， 三种血红素依赖性酪氨酸氧化酶的表征。每种酶都利用 一种单核血红素辅因子来氧化其酪氨酸基底物。有趣的是，细胞色素P450 来自结核分枝杆菌的蛋白质CYP 121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是一种新的 色氨酸双加氧酶超家族的成员，促进对 甲基化酪氨酸底物。过氧化物酶LmbB 2进行过氧化物酶型反应， 组氨酸而不是半胱氨酸的轴向配体。这些酶不仅催化基于酪氨酸的氧化 反应，但它们也与抗菌药物的开发有关。从血红素的相似性来看- 基于氧化剂和基板的结构，一个不可避免的问题出现，并将回答 这项研究是关于决定这些酶的催化活性的控制因素。在Aim中 #1，我们将确定CYP 121的机制和结构特征。使用电池 光谱和结构方法与合成探针相结合，我们将推出一种新的碳- 由P450酶介导的碳偶联机制。在目标#2中，我们将描述结构 和机制SfmD的重点是如何基板定位的铁结合的氧化剂 以及催化中间体的捕获。我们已经发现这种蛋白质是一种新的血红素- 氧合酶。目标#3将集中于研究由LmbB 2催化的过氧化物酶反应， 负责通过L-酪氨酸羟基化形成L-3，4-二羟基苯丙氨酸（L-DOPA）。我们 将利用小分子探针来询问机械途径。深入分析这些 三个相关的催化系统将测试我们的假设，关于血红素结合的氧化剂是如何 产生并导向芳香底物，解开结构-功能关系的 血红素酶的看似无关的超家族在更高的水平，并发展潜在的 进一步帮助合理的药物设计和发现过程的机制。

Abstract of the Parent Research Project One of the hallmarks of heme iron-dependent enzymes is the wide array of oxidation reactions that it can catalyze with its high-valent iron-oxo species. However, one conundrum is that each protein, in general, promotes only a specific type of reaction. How the reaction type is determined after the formation of the critical oxidant remains an open question whose answers have implications for the fundamental understanding of enzyme catalysis as well as de novo enzyme design and protein engineering. Because tyrosine is an essential building block of natural products, this project 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 of a more common hydroxylation reaction. We found that SfmD is a new member of the tryptophan dioxygenase superfamily that promotes a regioselective monooxygenation on 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, an inevitable question arises and will be answered by this study regarding the governing factors that determine the catalytic activity of these enzymes. In Aim #1, we will identify 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 an 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 will focus 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 pathways. 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.