Mechanism of biogenesis for built-in type quinone cofactor and application for composite-type catalytic antibody

内置型醌辅因子的生物发生机制及复合型催化抗体的应用

基本信息

批准号：
14560066
负责人：
OKAJIMA Toshihide
金额：
$ 2.62万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

To elucidate the biogenesis mechanism of built-in cofactors, the self-catalytic generation process of topaquinone cofactor (TPQ) was analyzed by frame-trapped X-ray crystallography, using copper-containing amine oxidase from Arthrobacter globiformis (AGAO). Then, X-ray structures of three intermediates during the TPQ biogenesis were determined, clearly showing the interactions with Cu^<2+> ion of precursor Tyr and its conformational charges during the biogenesis. Three tongued His residues coordinating Cu^<2+> ion were also substituted with Ala residue to clarify their roles on the TPQ biogenesis. The X-ray crystallographic and kinetic studies for these mutant enzymes demonstrate that precise position of Cu^<2+> ion is significantly important for the efficient TPQ biogenesis.To generate artificial quinone cofactor in the active site of AGAO, D298K mutant AGAO was prepared by site-directed mutagenesis. When D298K was activated by incubation with Cu^<2+> ion, a unique chromophore with λ_<***> of 450 nm, which is distinct from that of TPQ(λ_<***>=480 nm) in the wild type, was formed. By careful refinements in the X-ray crystallography of bolo D298K, it was found that C2 atom of TPQ ring is covalently bound to N_ε atom of Lys298 through imino double bond. Although the formation of lysine tyrosyl quinone would be expected, the identified quinone-like structure is a novel cofactor generated autocatalytically. Further, to produce a composite-type new quinone enzyme, Y382C mutant was produced. By incubating Y382C with mercaptophenol and Cu^<2+> ion, despite of undetectable UV/vis spectral changes, the low but apparent catalytic activity using phenethylamine was detected. Because no catalytic activity was detected in the absent of Cu^<2+>, it is possible that the biogenesis reaction forms any cofactor, resulting in the catalytic activity.

为了阐明内置辅因子的生物发生机制，使用框架捕获的X射线晶体学分析了甲喹酮辅因子（TPQ）的自催化生成过程，该过程使用含铜的球杆菌（Agao）中的含铜胺氧化物（AGAO）。然后，确定了TPQ生物发生过程中三个中间体的X射线结构，清楚地表明了与cu^<2+>离子的相互作用，在生物发生过程中与cu^<2+>离子的相互作用。三个舌，他的残留物协调的Cu^<2+>离子也受到ALA居住地，以阐明其在TPQ生物发生上的作用。这些突变酶的X射线晶体学和动力学研究表明，Cu^<2+>离子的精确位置对于有效的TPQ生物发生而言至关重要。在Agao中产生人造奎因酮辅助因子在AGAO的活性位置，由Site-Site d298K突变体Agao制备，由位置型突变进行制备。当D298K通过与Cu^<2+>离子孵育激活时，形成了一种与TPQ（λ_ <****> = 480 nm）在野生型中的独特发色团，与450 nm的λ_ <***>不同，在野生型中不同。通过在Bolo D298K的X射线晶体学中进行仔细的细化，发现TPQ环的C2原子通过免疫双键共价与Lys298的N_εAtom结合。尽管可以预期赖氨酸酪氨酸喹酮的形成，但鉴定出的喹酮样结构是一种新型的辅助因子产生的新型辅助因子。此外，为了产生复合型新奎因酶，生产了Y382C突变体。通过将Y382C与近载烯醇和Cu^<2+>离子一起孵育，尽管没有可检测到的UV/VIS光谱变化，则检测到使用苯乙烯乙胺的低但明显的催化活性。由于在没有Cu^<2+>的情况下未检测到催化活性，因此生物发生反应可能形成任何辅助因子，从而导致催化活性。