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一些独特的辅因子，这些辅因子来源于氨基酸残基，因此被称为内置辅因子，包括铜胺氧化酶的2,4,5-三羟基苯丙氨酸醌（topa醌；TPQ）和细菌胺脱氢酶的色氨酸色氨酸醌（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，为其分子设计提供依据。有代表性的成果如下。1)在反硝化副球菌和恶臭假单胞菌的醌类血红蛋白胺脱氢酶中发现了一种新的醌类辅助因子——半胱氨酸色氨酸醌（CTQ）。CTQ是继TPQ、TTQ和赖氨酸酪氨酸醌（LTQ）之后，第四个从氨基酸中提取的醌类辅助因子。2)利用大肠杆菌中过量产生的球形节杆菌重组铜胺氧化酶的无活性前体形式，我们证明了TPQ是通过酶蛋白的自我加工在结合铜离子的参与下产生的。3) x射线晶体学研究表明，低Cu/ tpq失活酶（脱酶）和含Cu/ tpq活性酶（全酶）仅在活性位点上存在结构差异。4)利用x射线晶体学研究了TPQ依赖铜的自氧化过程。载脂蛋白酶结晶用铜厌氧浸泡；从这种晶体中确定的结构提供了一种初始状态的观点：未修饰的酪氨酸与结合的铜配合。将铜结合晶体暴露在氧气中导致形成冻结的中间产物；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。这些是铜胺氧化酶中TPQ生物生成过程中第一个可视化的中间体。5)基于x射线晶体学和恒定活性位点碱基突变酶Asp298.6的动力学分析，提出了铜胺氧化酶的详细反应机理。一种假定的[Fe-S]结合蛋白已被证明是生物合成含CTQ的活性醌血红蛋白胺脱氢酶所必需的。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