Novel flavonol 2-oxoglutarate dependent dioxygenase: Affinity purification, characterization, and kinetic properties
Novel flavonol 2-oxoglutarate dependent dioxygenase: Affinity purification, characterization, and kinetic properties
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DOI：
10.1006/abbi.2000.2002
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发表时间：
2000-10-15
影响因子：
3.9
通讯作者：
Ibrahim, RK
中科院分区：
文献类型：
--
作者：
Anzellotti, D;Ibrahim, RK
一种依赖于2-氧戊二酸的双加氧酶[EC 1.14.11-]催化部分甲基化的黄酮醇的6-羟基化反应，已从美洲金藻中纯化到接近均一的水平。酶的纯化采用Superose 12和Mono Q柱层析，2-羟基戊二酸-琼脂糖亲和层析和免疫亲和层析。从MonoQ(97.1pkat/mg)中洗脱的6-羟基酶比活力提高了538倍，回收率为0.63%。这两个亲和层析步骤都能消除大多数污染蛋白质，但并不是没有酶活性和稳定性的损失。天然酶和变性酶的相对分子质量分别为42 kDa和45 kDa，提示为单体蛋白。该酶对具有7-甲氧基的部分甲基化黄酮醇的第6位表现出严格的专一性，表明它参与了该植物多甲基黄酮醇的生物合成。该酶的辅因子依赖性类似于其他植物双加氧酶，特别是它对亚铁离子催化活性和再激活的依赖性。内部氨基酸序列信息表明，它与其他植物黄酮类双加氧酶相关。底物相互作用动力学和产物抑制的结果。研究表明，这是一种有序的顺序反应机制(TERTER)，其中2-氧戊二酸是第一个结合的底物，其次是O-2和黄酮醇底物。产品的释放顺序相反，首先释放的是羟基黄酮醇，然后是二氧化碳和琥珀酸。据我们所知，这是第一个报道的依赖2-氧戊二酸的双加氧酶，它催化类黄酮化合物的芳香族羟基化。(C)2000年学术出版社。
A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition. studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O-2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound. (C) 2000 Academic Press.