Molecular Design of Composite Biocatalysts Containing Built-in Quinone Cofactor and Metals

近十年来，在各种酶蛋白中发现了许多源自氨基酸残基的独特辅因子，从而称为内置辅因子，包括铜胺氧化酶的2，4，5-三羟基苯丙氨酸醌（topa quinone; TPQ）和细菌胺脱氢酶的色氨酸甲萘醌（TTQ）。TPQ和TTQ都在酶基因中编码为普通氨基酸（Tyr或Trp），因此通过翻译后修饰合成。本研究旨在阐明含醌辅因子和金属的复合生物催化剂的结构和功能，为分子设计提供理论依据。本论文的主要研究成果如下：1）在副球菌和恶臭假单胞菌的醌血红素胺氧化酶中发现了一种新的醌辅因子--半胱氨酸醌（CTQ）。CTQ是继TPQ、TTQ和赖氨酸酪氨酰醌（LTQ）之后的第四种来源于氨基酸的醌辅因子。 ...更多信息 和赖氨酰氧化酶。2）使用在大肠杆菌中过量产生的来自球形节杆菌的重组铜胺氧化酶的无活性前体形式，我们证明了TPQ是在结合的铜离子的参与下通过酶蛋白的自加工产生的。3）Cu/TPQ-少失活酶的X射线晶体学研究（脱辅基酶）和含Cu/TPQ的活性形式（全酶）的结构差异仅在于活性位点。4）利用X射线晶体学，我们探索了TPQ的铜依赖性自氧化过程。载脂蛋白酶晶体厌氧浸泡与铜，从这个晶体确定的结构提供了一个视图的初始状态：未修饰的酪氨酸协调的结合铜。暴露的铜绑定晶体的氧气导致形成的冷冻捕获的中间体，结构分析表明，这些中间体含有二羟基苯丙氨酸醌和三羟基苯丙氨酸。这些是在铜胺氧化酶中TPQ生物发生过程中第一个可视化的中间体。5）基于对不变活性位点碱基Asp298的突变酶的X射线晶体学和动力学分析，提出了铜胺氧化酶的详细反应机理。6）推定的[Fe-S]结合蛋白已被证明是含CTQ的活性醌血红素蛋白胺脱氢酶的生物合成所必需的。少

Over the recent decade, a number of unique cofactors derived from amino acid residues and thereby called built-in cofactors have been discovered in various enzyme proteins, including 2,4,5-trihydroxyphenylalanine quinone (topa quinone ; TPQ) of copper amine oxidase and tryptophan tryptophylquinone (TTQ) of bacterial amine dehydrogenase. Both of TPQ and TTQ are encoded as ordinary amino acids (Tyr or Trp) in the enzyme genes and thus are synthesized by post-translational modification. The present studies aimed at elucidating the structure and function of composite biocatalysts containing built-in quinone cofactors and metals for their molecular design. Several representative achievements are as follows.1)We have identified a novel quinone cofactor, cysteine tryptophylquinone (CTQ) in quinohemoprotein amine dehydrogenases from Paracoccus denitrificans and Pseudomonas putida. CTQ is the forth quinone cofactor derived from amino acids, following TPQ, TTQ, and lysine tyrosylquinone (LTQ) fo … More und in lysyl oxidase.2)Using the inactive precursor forms of recombinant copper amine oxidases from Arthrobacter globiformis overproduced in Escherichia coli, we demonstrated that TPQ is generated through self-processing of the enzyme proteins with the participation of the bound copper ion.3)X-ray crystallographic studies of the Cu/TPQ-less inactive enzyme (apoenzyme) as well as the Cu/TPQ-containing active form (holoenzyme) revealed the structural difference only in the active site.4)Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ. Apo enzyme crystals were anaerobically soaked with copper ; the structure determined from this crystal provided a view of the initial state : the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates ; structural analyses indicated that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.5)A detailed reaction mechanism of copper amine oxidase has been proposed based on X-ray crystallographic and kinetic analyses of a mutant enzyme for the invariant active site base, Asp298.6)A putative [Fe-S]-binding protein has been shown to be essential for the biosynthesis of active quinohemoprotein amine dehydrogenase containing CTQ. Less