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.

Quercetinase是一种金属依赖的，共生成黄酮醇2，4-双加氧酶的酶，具有Cupin折叠。曲霉属(Aspergillusspp.)枯草芽孢杆菌和链霉菌的酶依赖于单核铜离子中心。菌株F1a可以利用不同的二价金属离子催化黄酮醇环的裂解。有趣的是，链霉菌的酶对Ni2+的活性最高，而Ni2+对芽孢杆菌酶来说是一个很差的辅因子。这种“金属混杂”的机制含义还没有得到很好的理解。拟议的项目将解决这个问题，使用不同金属形式的链霉菌quercetinase(QUED)作为模型酶。将分析金属离子对QUED的催化功能、活性中心几何形状和结构的影响：使用不同的黄酮醇底物，将对不同金属形式的QED和通过定点突变产生的QED变体进行动力学和电子顺磁共振波谱研究，以探讨(I)黄酮醇和/或O2与金属结合的模式(S)，(Ii)活性中心残基在催化中的可能作用，例如，作为一般碱，(Iii)可能的电子转移(从黄酮酸到双氧水？)。与H.Dobbek教授合作，将分析QUED形式的晶体结构。这个项目的总体目标是机械地比较金属依赖和辅因子非依赖的环裂解双加氧酶，以帮助理解双加氧酶是如何工作的。