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一些由氨基酸残基衍生的独特辅因子，包括铜胺氧化酶的2,4,5-三羟基苯基丙氨酸醌（topa醌；TPQ）和细菌胺脱氢酶的色氨酸色氨酸醌（TTQ）。这些醌辅因子暂时储存来自底物的还原等价物，然后将它们传递给外源的1-或2电子受体，如细胞色素、铜氧蛋白或分子氧。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。利用大肠杆菌中过量产生的球形节杆菌重组铜胺氧化酶的无活性前体形式，我们证明了TPQ是通过酶蛋白的自我加工在结合铜离子的参与下产生的。对Cu/TPQ-less失活酶…More（脱酶）和含Cu/ tpq - active形式（全酶）的x射线晶体学研究显示，它们的结构差异仅存在于活性位点。利用x射线晶体学研究了TPQ的铜依赖自氧化过程。载脂蛋白酶结晶用铜厌氧浸泡；从这种晶体中确定的结构提供了一种初始状态的观点：未修饰的酪氨酸与结合的铜配合。将铜结合晶体暴露在氧气中导致形成冻结的中间产物；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生物生成过程中第一个可视化的中间体。此外，我们最近在反硝化副球菌和恶臭假单胞菌的醌血红蛋白胺脱氢酶中发现了一种新的醌辅因子，半胱氨酸色氨酸醌（CTQ）。CTQ是继赖氨酸氧化酶中发现的TPQ、TTQ和赖氨酸酪氨酸醌（LTQ）之后，从氨基酸中衍生出的第四个醌辅因子。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