Anaerobic enzyme-substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase
Anaerobic enzyme-substrate structures provide insight into the reaction mechanism of the copper-dependent quercetin 2,3-dioxygenase
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DOI：
10.1073/pnas.262506299
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发表时间：
2002-12-24
影响因子：
11.1
通讯作者：
Dijkstra, BW
中科院分区：
文献类型：
--
作者：
Steiner, RA;Kalk, KH;Dijkstra, BW
槲皮素2,3-双加氧酶（2,3qd）是迄今为止唯一确定的铜双加氧酶。仅依靠单核Cu中心，它催化黄酮醇的o杂环断裂，产生更容易降解的酚类羧酸酯衍生物。在酶促过程中，两个C-C键断裂，同时释放一氧化碳。日本曲霉2,3qd与底物山奈酚和天然底物槲皮素厌氧配合的x射线结构分别在1.90和1.75埃分辨率下测定。黄酮醇通过3OH基团与铜离子配合成为单齿配体。它们占据靠近金属中心的一个浅而全面的疏水腔。由于最外向的黄酮醇a环与Pro（164）之间的范德华接触，活性位点前面的柔性环变得部分有序。有趣的是，与2,3qd结合的黄酮醇在C2原子上弯曲，这是金字塔化的。在这个原子上增加的局部sp(3)特征可以稳定碳中心自由基，使其被激活以进行双氧攻击。Glu（73）通过Oepsilon2原子与铜配位。其Oepsilon2原子与黄酮醇O3原子之间的距离约为2.55埃，表明两个原子之间存在氢键，表明Glu（73）可以作为黄酮醇去质子的碱，并保留了质子。基于结构的几何考虑表明O-2与黄酮醇C2原子结合是黄酮醇双氧化的首选途径。
Quercetin 2,3-dioxygenase (2,3QD) is the only firmly established copper dioxygenase known so far. Depending solely on a mononuclear Cu center, it catalyzes the breakage of the O-heterocycle of flavonols, producing more easily degradable phenolic carboxylic acid ester derivatives. In the enzymatic process, two C-C bonds are broken and concomitantly carbon monoxide is released, The x-ray structures of Aspergillus japonicus 2,3QD anaerobically complexed with the substrate kaempferol and the natural substrate quercetin have been determined at 1.90- and 1.75-Angstrom resolution, respectively. Flavonols coordinate to the copper ion as monodentate ligands through their 3OH group. They occupy a shallow and overall hydrophobic cavity proximal to the metal center. As a result of a van der Waals contact between the most outward flavonol A-ring and Pro(164), a flexible loop in front of the active site becomes partly ordered. Interestingly, flavonols bound to 2,3QD are bent at the C2 atom, which is pyramidalized. The increased local sp(3) character at this atom may stabilize a carbon-centered radical activated for dioxygen attack. Glu(73) coordinates the copper through its Oepsilon2 atom. The short distance of about 2.55 Angstrom between its Oepsilon2 atom and the flavonol O3 atom suggests that a hydrogen bond exists between the two atoms, indicating that Glu(73) can act as a base in flavonol deprotonation and that it retains the proton. Structure-based geometric considerations indicate O-2 binding to the flavonol C2 atom as the preferred route for flavonol dioxygenation.