Trapping of a dopaquinone intermediate in the TPQ cofactor biogenesis in a copper-containing amine oxidase from Arthrobacter globiformis
Trapping of a dopaquinone intermediate in the TPQ cofactor biogenesis in a copper-containing amine oxidase from Arthrobacter globiformis
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DOI：
10.1021/ja0731165
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发表时间：
2007-09-19
影响因子：
15
通讯作者：
Mure, Minae
中科院分区：
文献类型：
--
作者：
Moore, Robyn H.;Spies, M. Ashley;Mure, Minae
铜胺氧化酶（CAO）的托帕醌（TPQ）辅因子的生物合成是自催化的，并且需要铜和分子氧。已提出一种多巴醌中间体发生与铜相关的水分子的1,4 - 加成，以形成TPQ的还原形式（TPQ(red)），随后易被O₂氧化产生成熟的TPQ（TPQ(ox)）。在这项研究中，我们通过定点突变在球形节杆菌CAO（AGAO）的活性位点引入了一个赖氨酸残基，以产生D298K - AGAO。分辨率为1.7埃的D298K - AGAO的X射线晶体结构显示，在Lys298的ε - 氨基侧链和源自Tyr382（TPQ(ox)的前体）的一种多巴醌的C2位置之间形成了共价键。我们将该物质归为赖氨酸 - 酪氨酸醌（LTQ）的亚氨基醌互变异构体（LTI），LTQ是赖氨酸氧化酶（LOX）的有机辅因子。通过紫外/可见光谱和共振拉曼光谱对在pH 6.8时LTI形成的时间进程进行了追踪。在反应的早期阶段，观察到一种类似LTQ的中间体。然后该中间体以等吸光点的方式缓慢转化为LTI。不仅TPQ生物合成中多巴醌中间体的存在得到了证实，而且它也为在LOX中LTQ生物合成中所提出的多巴醌中间体的存在提供了有力支持。此外，这项研究表明AGAO中的多巴醌中间体是可移动的，并且能够从铜位点摆动到活性位点楔区与Lys298反应。
The biogenesis of the topaquinone (TPQ) cofactor of copper amine oxidase (CAO) is self-catalyzed and requires copper and molecular oxygen. A dopaquinone intermediate has been proposed to undergo 1,4-addition of a copper-associated water molecule to form the reduced form of TPQ (TPQ(red)), followed by facile oxidation by 02 to yield the mature TPQ (TPQ(ox)). In this study, we have incorporated a lysine residue in the active site of Arthrobacter globiformis CAO (AGAO) by site-directed mutagenesis to produce D298K-AGAO. The X-ray crystal structure of D298K-AGAO at 1.7-angstrom resolution revealed that a covalent linkage formed between the epsilon-amino side chain of Lys298 and the C2 position of a dopaquinone derived from Tyr382, a precursor to TPQox. We assigned the species as an iminoquinone tautomer (LTI) of lysine tyrosylquinone (LTQ), the organic cofactor of lysyl oxidase (LOX). The time course of the formation of LTI at pH 6.8 was followed by UV/vis and resonance Raman spectroscopies. In the early phase of the reaction, an LTQ-Iike intermediate was observed. This intermediate then slowly converted to LTI in an isosbestic manner. Not only is the presence of a dopaquinone intermediate in the TPQ biogenesis confirmed, but it also provides strong support for the proposed intermediacy of a dopaquinone in the biogenesis of LTQ in LOX. Further, this study indicates that the dopaquinone intermediate in AGAO is mobile and can swing from the copper site into the active-site wedge to react with Lys298.