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

近十年来，在不同的酶蛋白中发现了许多独特的氨基酸残基辅因子，包括铜胺氧化酶的2，4，5-三羟基苯丙氨酸醌(Topa Quone；TPQ)和细菌胺脱氢酶的色氨酸色氨酸(TTQ)。这些对苯二酚辅助因子在将来自底物的还原等价物传递给外源1电子或2电子受体(如细胞色素、铜还蛋白或分子氧)之前，会暂时存储它们。TPQ和TTQ都编码为酶基因中的普通氨基酸(Tyr或Trp)，因此都是通过翻译后修饰合成的。利用在大肠杆菌中过量生产的重组球形节杆菌铜胺氧化酶的非活性前体形式，我们证明了TPQ是通过结合铜离子参与酶蛋白的自我加工而产生的。铜/苯丙酮失活酶…的X射线结晶学研究More(脱辅酶)以及含铜/TPQ的活性形式(全酶)仅在活性部位显示了结构上的差异。用X射线结晶学方法研究了对苯二酚的铜依赖自氧化过程。APO酶晶体被铜厌氧浸泡；从这个晶体确定的结构提供了一个初始状态的视图：未修饰的酪氨酸与结合的铜配位。铜结合的晶体暴露在氧气中会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、苯二酚和三羟基苯丙氨酸。这些是铜胺氧化酶中TPQ生物合成过程中第一个可视化的中间体。此外，我们最近在脱氮副球藻和恶臭假单胞菌的藜红蛋白胺脱氢酶中发现了一种新的醌辅助因子--半胱氨酸色氨酸脱氢酶(CTQ)。CTQ是继TPQ、TTQ和赖氨酸酪氨酸醌(LTQ)之后，在赖氨酰氧化酶中发现的第四种由氨基酸衍生的醌辅因子。较少

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