Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

利用x射线衍射方法对反硝化副球菌的甲基胺脱氢酶（MADH）和芳香胺脱氢酶（AADH）的醌蛋白-电子转移体系进行结构研究。第一个系统涉及三种蛋白质，MADH， amicyanin和细胞色素c551i。MADH是124kda的H2L2二聚体，含有罕见的辅助因子色氨酸色氨酸醌（TTQ）。Amicyanin是一种12.5 kDa的蓝铜蛋白，与MADH特异性相互作用。细胞色素c551i是一种分子量为17.5 kDa的蛋白质，它可以接受来自氨基青素的电子，并通过另一种细胞色素将它们转移到末端氧化酶。测定了MADH、花青素、MADH与花青素的二元配合物以及MADH、花青素与细胞色素c551i的三元配合物的结构。AADH是114kda的H2L2异四聚体，也含有TTQ。它的电子受体是蓝铜蛋白，一种15 kDa的蓝铜蛋白。天然配合物的结构将在高分辨率下进行细化，二元和三元配合物的几种氧化还原变体将进行分析。我们将研究这些复合物中amicyanin和MADH的突变，以分析这些突变引起的结构变化。MADH及其电子转移配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。我们将完成从Methylophilus W3A1中提纯两种天然形式和两种抑制形式的MADH，它们使用细胞色素而不是花青素作为主要的电子受体。此外，还将对另外两种生物，即勒索甲基杆菌AM1和鞭毛甲基杆菌KT进行一系列的MADH研究。最后，将确定AADH的结构及其与azurin的推测复合物，并与副球菌酶进行比较。电子转移是许多生物过程的基础，但由于其组分通常不溶且不能结晶，因此很难从结构上进行研究。对MADH和AADH酶系统的研究将为这一过程提供有价值的信息。这种可溶性电子传递系统的各种组分的相互作用确定了识别和控制电子传递的重要分子特征，并确定了电子流的可能途径。提出的突变和配体诱导的MADH系统的扰动将进一步探讨这一复杂的过程。此外，TTQ辅助因子氧化底物的分子细节将揭示该辅助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助因子有何不同。TTQ是不寻常的，因为它是直接从基因组DNA编码的两个氨基酸侧链融合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.