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

近十年来，在各种酶蛋白中发现了许多独特的氨基酸残基衍生的辅因子，包括铜胺氧化酶的2，4，5-三羟基苯丙氨酸醌（topa quinone; TPQ）和细菌胺脱氢酶的色氨酸甲萘醌（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性1-或2-电子受体，如细胞色素、铜氧还蛋白或分子氧。TPQ和TTQ都在酶基因中编码为普通氨基酸（Tyr或Trp），因此通过翻译后修饰合成。使用无活性的前体形式的重组铜胺氧化酶从球形节杆菌在大肠杆菌中过度生产，我们表明，TPQ是通过自我加工的酶蛋白质的参与结合的铜离子。Cu/TPQ非活性酶的X射线晶体学研究 ...更多信息 （脱辅基酶）以及Cu/TPQ含活性形式（全酶）揭示了结构差异，只有在活性位点。用X射线晶体学方法研究了TPQ的铜依赖自氧化过程。载脂蛋白酶晶体厌氧浸泡与铜，从这个晶体确定的结构提供了一个视图的初始状态：未修饰的酪氨酸协调的结合铜。暴露的铜绑定晶体的氧气导致形成的冷冻捕获的中间体，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，我们最近已经确定了一种新的醌辅因子，半胱氨酸醌（CTQ）的醌血红素蛋白胺脱氢酶从副球菌和恶臭假单胞菌。CTQ是继TPQ、TTQ和赖氨酰氧化酶中发现的赖氨酸酪氨酰醌（LTQ）之后的第四种来源于氨基酸的醌辅因子。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