Metall-Spezifität und Katalysemechanismus der Quercetinase QueD

槲皮素酶QueD的金属特异性及催化机制

• 批准号: 163739897
• 负责人: Professorin Dr. Susanne Fetzner
• 金额: --
• 依托单位: Institut für Molekulare Mikrobiologie und Biotechnologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/163739897?language=en
• 关键词: Metall; Spezifit; und; Katalysemechanismus; der

Quercetinases are metal-dependent, CO-forming flavonol 2,4-dioxygenases with a cupin fold. Whereas the reactivity of quercetinases of Aspergillus spp. depends on a mononuclear Cu(II) center, the enzymes from Bacillus subtilis and Streptomyces sp. strain FLA can use different divalent metal ions to catalyze flavonol ring cleavage. Interestingly, the Streptomyces enzyme is most active with Ni2+, which is a poor cofactor for the Bacillus enzyme. The mechanistic implications of such “metal promiscuity” are not well understood. The proposed project will address this question, using different metal forms of Streptomyces quercetinase (QueD) as model enzyme. The effect of metal ions on catalytic function, active-site geometry, and structure of QueD will be analyzed: Using different flavonol substrates, kinetic and electron paramagnetic resonance spectroscopic studies will be performed on different metal forms of QueD and on QueD variants generated by site-directed mutagenesis, to address (i) the mode of flavonol and/or O2 binding to the metal(s), (ii) the possible role of active-site residues in catalysis, e.g., as a general base, (iii) possible electron transfer (from flavonolate to dioxygen?). In collaboration with Prof. Dr. H. Dobbek, the crystal structure of QueD forms will be analyzed. The overall goal of this project is to mechanistically compare metal-dependent and cofactor-independent ring-cleavage dioxygenases, to contribute to an understanding of how dioxygenases work.

槲皮素酶是金属依赖性的，共同形成的黄酮醇2,4-二加氧酶，具有up折。而曲霉属的槲皮素的反应性。取决于单核Cu（II）中心，来自枯草芽孢杆菌和链霉菌的酶。 FLA FLA可以使用不同的二价金属离子来催化黄酮醇环的裂解。有趣的是，链霉菌酶对Ni2+的活性最活跃，Ni2+是芽孢杆菌酶的较差的辅助因子。这种“金属承诺”的机械含义尚不清楚。拟议的项目将使用不同的金属形式的链霉菌槲皮素酶（qued）作为模型酶来解决这个问题。将分析金属离子对催化功能，活性位点几何形状和Qued结构的影响：使用不同的黄酮底物，动力学和电子磁性共振光谱研究将对QUED的不同金属形式进行，并在QUED的不同金属形式上对被位置为代码的固定型（II）模式（i）模式（i）属于flavonol of Flavovonol和/o）。活性位点在催化剂中的可能作用，例如，作为一般碱，（iii）可能的电子转移（从黄酮盐到二恶英？）。与H. Dobbek博士合作，将分析要求形式的晶体结构。该项目的总体目标是将机械学比较金属依赖性和与辅助因子无关的环状裂解二氧酶，以有助于理解对二氧酶的工作方式。