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)-reticuline氧化环化为(S)-scoulerine，两者都是植物苯基异喹啉生物碱生物合成途径中的中间体。属于这个家族的生物碱，包括小檗碱、棕榈碱和血碱，已经证实具有抗菌和抗癌活性。BBE催化的区域选择性环化反应是新颖的；只有另外两种黄酮酶被证明能催化类似的反应，而且这种环化还没有被合成的有机方法复制。本项目的目标是酶活性的机制分析，包括区分逐步和协调机制，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，以及原生和溶剂动力学同位素效应，以确定与be催化反应相关的单个速率常数，并确定速率决定步骤。活性位点残基的作用将通过位点定向诱变研究来探讨。

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.