Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

9728885用X-射线衍射法对脱氮拟球藻的藜麦蛋白-电子转移系统甲胺脱氢酶和芳香胺脱氢酶进行了结构研究。第一个系统涉及三个蛋白质：MADH、氨基花青素和细胞色素C551I。MADH是一种大小为124 kDa的H_2二聚体，含有不寻常的辅因子色氨酸-色苯二酚(TTQ)。氨蓝蛋白是一种大小为12.5 kDa的蓝铜蛋白，能与MADH特异结合。细胞色素C551I是一种17.5 kDa的蛋白质，它可以接受氨蓝蛋白的电子，并通过另一种细胞色素将电子转移到末端的氧化酶上。测定了MADH、氨基花青素、MADH与氨基花青素的二元络合物以及MADH、氨基花青素和细胞色素C551I的三元络合物的结构。AADH是一种114 kDa的H_2L2杂四聚体，也含有TTQ。它的电子受体是天青素，一种15 kDa的蓝铜蛋白。天然络合物的结构将以高分辨率进行精炼，并将分析二元和三元络合物的几种氧化还原变体的结构。将研究复合体中氨基花青素和MADH的突变，以分析这些突变引起的结构变化。MADH及其电子转移络合物的研究也将在与底物和抑制剂反应后以及在不同的氧化还原状态下进行。将完成从甲嗜甲基菌W3A1提纯两种天然形式和两种被抑制形式的MADH，它使用细胞色素而不是氨基花青素作为主要电子受体。此外，还将对另外两种生物--甲基鞭毛杆菌AM1和鞭毛杆菌KT的MADH进行一系列研究。最后，将确定AADH及其与天青素的假定络合物的结构，并与Paracoccus酶进行比较。电子转移是许多生物过程的基础，但很难从结构上进行研究，因为这些成分通常是不溶的，也不能结晶。对MADH和AADH酶系统的拟议研究将提供关于这一过程的有价值的信息。这一可溶电子传输系统的各种成分的相互作用定义了对识别和控制电子传输非常重要的分子特征，并定义了电子流动的可能路径。所提出的MADH系统的突变和配体诱导的扰动将进一步探索这一复杂的过程。此外，TTQ辅因子氧化底物的分子细节将有助于阐明该辅因子如何在体内发挥作用，以及它与其他更常见的氧化还原辅因子有何不同。TTQ是不同寻常的，因为它是由基因组DNA编码的两个氨基酸侧链直接融合而成，而不是通过单独的生物合成途径获得的。MADH和AADH系统非常适合在分子水平上提供对这些重要过程的理解。

9728885 Mathews Structural studies will be carried out on the quinoprotein-electron transfer systems methylamine dehydrogenase (MADH) from Paracoccus denitrificans and aromatic amine dehydrogenase (AADH) by the methods of x-ray diffraction. Three proteins are involved in the first system, MADH, amicyanin and cytochrome c551i. MADH is an H2L2 dimer of 124 kDa and contains the unusual cofactor tryptophan tryptophylquinone (TTQ). Amicyanin is a blue copper protein of 12.5 kDa which interacts specifically with MADH. Cytochrome c551i is a 17.5 kDa protein which can accept electrons from amicyanin and transfer them via another cytochrome to a terminal oxidase. The structures of MADH, amicyanin, the binary complex of MADH with amicyanin and the ternary complex of MADH, amicyanin and cytochrome c551i have been determined. AADH is an H2L2 heterotetramer of 114 kDa and also contains TTQ. Its electron acceptor is azurin, a blue copper protein of 15 kDa. Structures of the native complexes will be refined at high resolution and those of several redox variants of the binary and ternary complexes will be analyzed. Mutants of amicyanin and of MADH in the complexes will be studied to analyze the structural changes caused by these mutations. Studies of MADH and its electron transfer complexes will also be carried out after reaction with substrates and inhibitors and in different redox states. The refinement of two native and of two inhibited forms of MADH from Methylophilus W3A1, which uses a cytochrome rather than amicyanin as the primary electron acceptor, will be completed. A series of studies of MADH from two other organisms, Methylobacterium extorquens AM1 and Methylobacillus flagellatum KT will also be studied. Finally, the structure of AADH and its putative complex with azurin will be determined and compared with the Paracoccus enzyme. Electron transfer is fundamental to many biological processes, but is difficult to study structurally since the components are usually insoluble and cannot b e crystallized. The proposed studies of the MADH and AADH enzyme systems will provide valuable information about this process. The interaction of the various components of this soluble electron transport system define molecular features important for recognition and control of electron transfer and define likely pathways for electron flow. The proposed mutational and ligand-induced perturbations of the MADH system will further probe this complex process. In addition, the molecular details of substrate oxidation by the TTQ cofactor will shed light on how this cofactor functions in vivo and how it differs from other, more common redox cofactors. TTQ is unusual because it is obtained directly from the fusion of two amino acid side chains coded by genomic DNA rather than from a separate biosynthetic pathway. The MADH and AADH systems are well suited to provide an understanding of these important processes at the molecular level.