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

为了阐明内置协因子的生物发生机制，以全球节杆菌(Arthrobacter Globbiformis，AGAO)含铜胺氧化酶为研究对象，用X-射线衍射法分析了自催化生成TOPQ的过程。然后，测定了三种中间体在TPQ生物发生过程中的X射线结构，清楚地显示了生物发生过程中前驱体Tyr与Cu2+&lt；2+&gt；离子及其构象电荷的相互作用。此外，还用丙氨酸残基取代了三个与铜离子配位的舌状His残基，以阐明它们在TPQ生物发生中的作用。对这些突变酶的X-射线晶体结构和动力学研究表明，准确的Cu2+和gt；离子位置对于高效的TPQ生物合成是非常重要的。为了在agao的活性部位产生人工醌辅助因子，通过定点突变获得了D298K突变体agao。当D298K与Cu2+&gt；离子孵育被激活时，形成了一个独特的发色团，其λ_&lt；*&gt；为450 nm，与野生型Tpq的λ_&lt；*&gt；=480 nm不同。通过对BLO D298K晶体结构的改进，发现TPQ环上的C2原子与LYS298上的N_ε原子以亚氨基双键共价键合。虽然赖氨酸酪氨酸苯二酚的形成是预期的，但所鉴定的类苯醌结构是一种新的自催化产生的辅因子。此外，为了产生一种复合型新的苯二酚酶，还产生了Y382C突变体。通过将Y382C与硫代苯酚和Cu2+&Gt；离子反应，尽管没有检测到UV/Vis光谱的变化，但对苯乙胺的催化活性很低，但却很明显。由于在没有Cu2+的情况下没有检测到催化活性，因此生物发生反应有可能形成任何辅因子，从而导致催化活性。

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.