Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

9419899马修斯用X-射线衍射方法对副球菌（Paracoccus）的醌蛋白-电子传递系统甲胺脱氢酶（MADH）进行了结构研究。 三种蛋白质参与该系统，MADH，amicyanin和细胞色素c551 i。 MADH是一种124 kDa的H2 L2二聚体，含有不常见的辅因子色氨酸甲萘醌（TTQ）。 Amicyanin是一种分子量为12.5 kDa的蓝铜蛋白，与MADH特异性相互作用。 细胞色素c551 i是一种17.5 kDa的蛋白质，它可以接受来自amicyanin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、amicyanin、MADH与amicyanin的二元复合物以及MADH、amicyanin与细胞色素c551 i的三元复合物的结构。 通过低温冷却和同步辐射的使用，本络合物的精制将扩展到更高的分辨率。 amicyanin的突变体将单独和在复合物中进行研究。 MADH及其电子转移复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。 来自嗜甲基菌W3 A1的MADH的结构，其使用细胞色素而不是amicyanin作为主要电子受体，将被完成并与副球菌酶进行比较。 最后，将尝试使W3 A1酶与来自相同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，但很难从结构上研究，因为这些成分通常不溶，不能结晶。 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种组分的相互作用定义了对于识别和控制电子传递重要的分子特征，并定义了电子流动的可能途径。 提出的突变和配体诱导的系统扰动将进一步探索这一复杂的过程。 此外，底物氧化的TTQ辅因子的分子细节将阐明如何在体内的辅因子的功能，以及它如何不同于其他更常见的氧化还原辅因子。 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的融合而不是从单独的生物合成途径获得的。 MADH系统非常适合于在分子水平上提供对这些重要过程的理解。 ***

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. ***