Mechanism of Berberine Bridge Enzyme

描述（由申请人提供）：小檗碱桥酶（BBE）是一种含黄素的氧化酶，可催化（S）-牛心果碱氧化环化为（S）-臭牡丹碱，这两种中间体都是植物中苄基异喹啉生物碱生物合成途径的中间体。属于该家族的生物碱，包括小檗碱、巴马汀和血根碱，已证实具有抗菌和抗癌活性。由BBE催化的区域选择性环化反应是新颖的;只有两种其他的黄素酶被证明可以催化类似的反应，并且这种环化还没有被合成有机方法复制。本项目的目标是酶活性的机理分析，包括分步和协同机制之间的区别，并确定活性位点残基的作用。这些研究将涉及稳态和快速反应动力学，以及初级和溶剂动力学同位素效应，以确定与BBE催化反应相关的个别速率常数，并确定速率决定步骤。活性位点残基的作用将通过定点诱变研究进行探索。 公共卫生相关性：该补助金的主要目的是为海伦娜·加韦斯卡（Helena Gaweska）的博士后培训提供支持，为多元化的训练有素的科学家人才库做出贡献，以满足国家的生物医学研究需求。被研究的酶，黄连素桥酶，催化生物合成具有抗菌和抗癌活性的生物碱的一个不寻常的步骤。对其机制的进一步了解可能会导致新药物的合成方法。

DESCRIPTION (provided by applicant): Berberine Bridge Enzyme (BBE) is a flavin-containing oxidase that catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, both intermediates in the benzylisoquinoline alkaloid biosynthesis pathways in plants. Alkaloids belonging to this family, including berberine, palmatine, and sanguinarine, have confirmed antimicrobial and anticancer activities. The regioselective cyclization reaction catalyzed by BBE is novel; only two other flavoenzymes have been shown to catalyze similar reactions, and this cyclization has not yet been replicated by synthetic organic methods. The goal of this proposed project is mechanistic analysis of the enzyme activity, including differentiation between stepwise and concerted mechanisms, and determination of the roles of active site residues. These studies will involve steady-state and rapid-reaction kinetics, as well as primary and solvent kinetic isotope effects, in order to determine individual rate constants associated with the BBE-catalyzed reaction and to identify rate-determining steps. The roles of active site residues will be probed by site-directed mutagenesis studies. PUBLIC HEALTH RELEVANCE: The primary purpose of this grant is to provide support for the postdoctoral training of Helena Gaweska to contribute to a diverse pool of highly-trained scientists available to address the Nation's biomedical research needs. The enzyme to be studied, berberine bridge enzyme, catalyzes an unusual step in the biosynthesis of alkaloids with antimicrobial and anticancer activities. Increased understanding of its mechanism may lead to synthetic approaches to novel drugs.