Mechanism of Berberine Bridge Enzyme

描述(申请人提供)：小檗碱桥酶(BBE)是一种含黄素的氧化酶，催化(S)-网状生物碱氧化环化为(S)-黄连素，这两个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，包括黄连素、巴马汀和血根碱，已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一种新颖的反应，只有两种黄素酶被证明能催化类似的反应，这种环化反应还没有被合成的有机方法复制。这个拟议项目的目标是对酶的活性进行机械分析，包括区分分步和协调机制，并确定活性部位残基的作用。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催化反应有关的各个速率常数，并确定决定速率的步骤。活性位点残基的作用将通过定点突变研究来探索。

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.