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.

为了阐明内置辅助因子的生物发生机制，使用含铜的胺氧化酶从含量的球杆菌（AGAO）（agao），然后确定了三个中间体的X射线结构，清楚地显示了X射线结构，清楚地显示了X射线结构。在生物发生过程中，与Cu^<2+> s构象的相互作用。 Cu^<2+>离子的精确位置对于TPQ生物发生非常重要。要在Agao +> ion的活性位点生成人造醌辅助因子，这是一种独特的发色团，具有450 nm的λ_<***>与野生型中的TPQ（λ_ <***> = 480 nm）不同，这是通过在Bolo D298K的X射线晶体学中仔细的细化而形成的。通过Imino双键的N_ε原子。 +>离子，尽管在没有Cu^<2+>的情况下未检测到使用苯乙胺的紫外线/vis光谱变化，但在没有Cu^<2+>的情况下检测到了催化作用，但生物发生反应可能形成任何辅助反应。