Structure, Catalytic Function and Biogenesis Mechanism of Novel Built-in Quinone Cofactors

新型内置醌辅因子的结构、催化功能和生物发生机制

基本信息

批准号：
12480180
负责人：
TANIZAWA Katsuyuki
金额：
$ 10.05万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

Over the recent decade, a number of unique cofactors derived from amino acid residues have been discovered in various enzyme proteins, including 2,4,5-trihydroxyphenylalanine quinone (topa quinone; TPQ) of copper amine oxidase and tryptophan tryptophylquinone (TTQ) of bacterial amine dehydrogenase. These quinone co factors temporarily store the reducing equivalents derived from the substrate before passing them on to exogenous 1- or 2-electron acceptors such as a cytochrome, a cupredoxin, or molecular oxygen. Both of TPQ and TTQ are encoded as ordinary amino acids (Tyr or Trp) in the enzyme genes and thus are synthesized by post-translational modification. Using the inactive precursor forms of recombinant copper amine oxidases from Arthrobacter globiformis overproduced in Escherichia coli, we demonstrated that TPQ is generated through self-processing of the enzyme proteins with the participation of the bound copper ion. X-ray crystallographic studies of the Cu/TPQ-less inactive enzyme … More (apoenzyme) as well as the Cu/TPQ-containing active form (holoenzyme) revealed the structural difference only in the active site. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provided a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicated that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase. Furthermore, we have recently identified a novel quinone co factor, cysteine tryptophylquinone (CTQ) in quinohemoprotein amine dehydrogenases from Paracoccus denitrificans and Pseudomonas putida. CTQ is the forth quinone co factor derived from amino acids, following TPQ, TTQ, and lysine tyrosylquinone (LTQ) found in lysyl oxidase. Less

近十年来，在各种酶蛋白中发现了许多源自氨基酸残基的独特辅因子，包括铜胺氧化酶的2,4,5-三羟基苯丙氨酸醌（topa quinone；TPQ）和细菌的色氨酸色氨酸醌（TTQ）这些醌辅因子暂时储存来自底物的还原当量，然后将其传递给外源 1- 或2-电子受体，例如细胞色素、铜氧还蛋白或分子氧，TPQ和TTQ均被编码为酶基因中的普通氨基酸（Tyr或Trp），因此使用非活性前体通过翻译后修饰合成。通过在大肠杆菌中过量产生球形节杆菌的重组铜胺氧化酶形式，我们证明了 TPQ 是通过自我加工产生的在结合铜离子的参与下，对不含 Cu/TPQ 的无活性酶（脱辅基酶）以及含 Cu/TPQ 的活性形式（全酶）进行 X 射线晶体学研究揭示了结构差异。仅在活性位点，我们探测了 TPQ 酶晶体的铜依赖性自动氧化过程，并确定了铜的结构。该晶体提供了初始状态的视图：未修饰的酪氨酸与结合的铜配位，将铜结合的晶体暴露于氧气导致形成冷冻捕获的中间体；结构分析表明这些中间体含有二羟基苯丙氨酸醌和三羟基苯丙氨酸。这些是铜胺氧化酶 TPQ 生物发生过程中第一个可视化的中间体。此外，我们最近还发现了一种新的醌辅因子，半胱氨酸。脱氮副球菌和恶臭假单胞菌的醌血红蛋白胺脱氢酶中的色氨酸醌 (CTQ) 是继赖氨酰氧化酶中的 TPQ、TTQ 和赖氨酸酪氨醌 (LTQ) 之后的第四种源自氨基酸的醌辅因子。