Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

醌蛋白电子转移系统的晶体学研究：甲胺脱氢酶

• 批准号: 9419899
• 负责人: F. Mathews
• 金额: $ 40.5万
• 依托单位: Washington University School of Medicine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-03-01 至 1998-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9419899&HistoricalAwards=false
• 关键词: Crystallographic; Study; Quinoprotein; Electron; Transfer

9419899 Mathews Structural studies will be carried out on the quinoprotein-electron transfer system methylamine dehydrogenase (MADH) from Paracoccus denitrificans by the methods of x-ray diffraction. Three proteins are involved in this 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. Refinement of the present complexes will be extended to higher resolution by cryo-cooling and use of synchrotron radiation. Mutants of amicyanin will be studied both alone and in the complexes. 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 structure of MADH from Methylophilus W3A1, which uses a cytochrome rather than amicyanin as the primary electron acceptor, will be completed and compared with the Paracoccus enzyme. Finally, attempts will be made to co-crystallize the W3A1 enzyme with a cytochrome electron acceptor from the same organism. %%% Electron transfer is fundamental to many biological processes, but is difficult to study structurally since the components are usually insoluble and cannot be crystallized. The proposed studies of the MADH enzyme system will provide valuable information about this process. The interaction of the three 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 perturbati ons of the system will further probe this complex precess. 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 system is well suited to provide an understanding of these important processes at the molecular level. ***

用x射线衍射方法对反硝化副球菌的醌蛋白-电子转移体系甲胺脱氢酶（MADH）进行了结构研究。该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。通过低温冷却和同步辐射的使用，将现有复合物的改进扩展到更高的分辨率。将单独研究和在复合物中研究花青素的突变体。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。将完成来自Methylophilus W3A1的MADH的结构，并将其与副球菌酶进行比较，该酶使用细胞色素而不是amyyanin作为主要电子受体。最后，将尝试将W3A1酶与来自同一生物体的细胞色素电子受体共结晶。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三个组成部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系统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系统非常适合在分子水平上提供对这些重要过程的理解。＊＊＊