EPR characterization of the mononuclear Cu-containing Aspergillus japonicus quercetin 2,3-dioxygenase reveals dramatic changes upon anaerobic binding of substrates
EPR characterization of the mononuclear Cu-containing Aspergillus japonicus quercetin 2,3-dioxygenase reveals dramatic changes upon anaerobic binding of substrates
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DOI：
10.1046/j.1432-1033.2002.02973.x
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发表时间：
2002-06-01
期刊：
影响因子：
--
通讯作者：
Huber, M
中科院分区：
文献类型：
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作者：
Kooter, IM;Steiner, RA;Huber, M
槲皮素2，3-双加氧酶（2，3QD）是一种含铜的双加氧酶，其催化黄酮醇槲皮素氧化为2-原儿茶酰基间苯三酚羧酸，并伴随产生一氧化碳。与铁双加氧酶相反，对铜双加氧酶知之甚少。本文用电子顺磁共振谱（EPR）对曲霉菌2，3-QD进行了表征。在pH 6.0时，2，3QD显示两种EPR物质的混合物。主要形式具有2型Cu位点的典型参数（g（//）= 2.330，A（//）= 13.7 mT），次要形式具有更扭曲的几何形状（g（//）= 2.290，A（//）= 12.5 mT）。底物槲皮素的厌氧添加导致不同的单一物种EPR谱，其中g（//）= 2.336，A（//）= 11.4mT，参数在Peisach-Blumberg（g（//）vs. A（//））图中介于2型和1型Cu位点的参数之间。翻转后，观察到新的EPR信号，这归因于羧酸酯产物复合物。该光谱是类似的天然酶在pH 10.0，并具有g -张量参数表明三角双锥网站。在所研究的各种黄酮类化合物中，只有黄酮醇能够与2，3QD的铜中心结合。分析了9种在A环和B环上具有不同羟基化模式的黄酮醇。它们聚集在Peisach-Blumberg图的两个不同区域，并表明5-OH基团的存在对A（//）参数有很大的影响。注意到A. B12，3QD和A. 821.第821章我的世界
Quercetin 2,3-dioxygenase (2,3QD) is a copper-containing dioxygenase that catalyses the oxidation of the flavonol quercetin to 2-protocatechuoylphloroglucinol carboxylic acid with concomitant production of carbon monoxide. In contrast to iron dioxygenases, very little is known about copper dioxygenases. We have characterized 2,3QD from the fungus Aspergillus japonicus by electron paramagnetic resonance spectroscopy (EPR). At pH 6.0, 2,3QD shows a mixture of two EPR species. The major form has parameters typical of type 2 Cu sites (g (//) = 2.330, A (//) = 13.7 mT), the minor one has a more distorted geometry (g (//) = 2.290, A (//) = 12.5 mT). Anaerobic addition of the substrate quercetin results in a different, single species EPR spectrum with g (//) = 2.336, A (//) = 11.4 mT, parameters, which are in-between those of the type 2 and type 1 Cu sites in the Peisach-Blumberg (g (//) vs. A (//) ) plot. After turnover, a new EPR signal is observed, which is ascribed to the carboxylic acid ester product complex. This spectrum is similar to that of the native enzyme at pH 10.0 and has g -tensor parameters suggesting a trigonal bipyramidal site. Of a variety of flavonoids studied, only flavonols are able to bind to the copper centre of 2,3QD. Nine flavonols with different hydroxylation patterns at the A- and B-ring have been analysed. They cluster in two different regions of the Peisach-Blumberg plot and show that the presence of a 5-OH group has a large effect on the A (//) parameter. Several differences are noted between A. japonicus 2,3QD and the enzyme from A. niger German Collection of Microorganisms 821.