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

为了阐明内建辅因子的生物发生机制，利用球形节杆菌（Arthrobacter globiformis， AGAO）含铜胺氧化酶，采用框架捕获x射线晶体学分析了topaquinone辅因子（TPQ）的自催化生成过程。然后，测定了TPQ生物生成过程中三个中间体的x射线结构，清楚地显示了TPQ生物生成过程中前体Tyr与Cu^<2+>离子的相互作用及其构象电荷。3个配位Cu^<2+ b>离子的舌形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。这些突变酶的x射线晶体学和动力学研究表明，Cu^<2+ b>离子的精确位置对TPQ的有效生物生成至关重要。为了在AGAO活性位点产生人工醌辅助因子，采用定点诱变法制备了D298K突变体AGAO。当D298K与Cu^<2+>离子孵育后，形成了与野生型TPQ（λ_<***>=480 nm）不同的λ <***>为450 nm的独特发色团。通过对bolo D298K的x射线晶体学的仔细改进，发现TPQ环的C2原子通过亚氨基双键与Lys298的N_ε原子共价结合。虽然赖氨酸酪氨酸醌的形成是预期的，但所鉴定的类醌结构是一种新的自催化生成的辅助因子。在此基础上，通过Y382C突变体制备复合型醌酶。通过与巯基苯酚和Cu^<2+>离子孵育Y382C，尽管紫外/可见光谱变化不明显，但苯乙胺对Y382C的催化活性较低，但明显。由于在Cu^<2+>缺失的情况下没有检测到催化活性，因此可能是生物生成反应形成了任何辅助因子，导致了催化活性。

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.