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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.

为了阐明内建辅因子的生物合成机制，采用框捕获X射线晶体学方法，以球形节杆菌（Arthrobacter globiformis，AGAO）的含铜胺氧化酶为催化剂，研究了托派醌辅因子（topaquinone cofactor，TPQ）的自催化合成过程.测定了TPQ生物合成过程中三个中间体的X射线结构，清晰地显示了TPQ生物合成过程中前体Tyr与Cu^2+离子及其构象电荷的相互作用。三个配位Cu^<2+>的舌形His残基也被Ala残基取代，以阐明它们在TPQ生物发生中的作用。为了在AGAO的活性位点合成人工醌辅因子，采用定点突变法制备了D298 K突变体AGAO。当D298 K被Cu^<2+>激活后，形成了一个独特的发色团，其λ <*>为450 nm，这与野生型TPQ（λ <*>=480 nm）不同。通过对博洛D298 K的X射线晶体学研究，发现TPQ环上的C2原子与Lys 298的N_ε原子通过亚氨基双键共价结合。虽然赖氨酸酪氨酰醌的形成是预期的，但鉴定的醌样结构是自催化产生的新型辅因子。此外，为了生产复合型新醌酶，生产了Y382 C突变体。通过将Y382 C与巯基酚和Cu^2+离子一起孵育，尽管检测不到UV/维斯光谱变化，但检测到使用苯乙胺的低但明显的催化活性。由于在不存在Cu^<2+>的情况下没有检测到催化活性，因此可能是生物发生反应形成了任何辅因子，从而导致了催化活性。

项目成果

期刊论文数量（52）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

X-ray snapshots of quinone cofactor biogenesis in bacterial copper amine oxidase

DOI：
10.1038/nsb824
发表时间：
2002-08-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
0
作者：
Kim, M;Okajima, T;Yamaguchi, H
通讯作者：
Yamaguchi, H

タンパク質科学(後藤祐児・桑島邦博・谷澤克行編)

蛋白质科学（后藤雄二、桑岛邦宏、谷泽克之编）

DOI：
发表时间：
2005
期刊：
影响因子：
0
作者：
岡島俊英;谷澤克行
通讯作者：
谷澤克行

岡島俊英, 谷澤克行: "ペプチド・ビルトイン型キノン補酵素研究の新展開"蛋白質核酸酵素. 48. 740-746 (2003)

Toshihide Okajima、Katsuyuki Tanizawa：“肽和内置醌辅酶研究的新进展”蛋白质核酸酶 48. 740-746 (2003)。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Scanning tunnelling microscopy images of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms adsorbed on gold under ambient conditions

DOI：
10.1143/jjap.41.3916
发表时间：
2002-06-01
期刊：
JAPANESE JOURNAL OF APPLIED PHYSICS PART 1-REGULAR PAPERS BRIEF COMMUNICATIONS & REVIEW PAPERS
影响因子：
0
作者：
Contera, SA;Okajima, T;Iwasaki, H
通讯作者：
Iwasaki, H

ペプチド・ビルトイン型キノン補酵素の生成磯構

肽内置醌辅酶的生产结构