STRUCTURAL STUDIES OF COMPLEX IRON-SULFUR FLAVOPROTEINS

复杂铁硫黄素蛋白的结构研究

• 批准号: 3279758
• 负责人: F SCOTT MATHEWS
• 金额: $ 11.45万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-01-01 至 1988-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3279758
• 关键词: X ray crystallography; computer graphics /printing; enzyme model; enzyme structure; flavoproteins; iron; metalloenzyme; sulfur compounds

The molecular structure of Trimethylamine Dehydrogenase (TMADH) from the methylotrophic bacterium W3A1 will be studied by X-ray diffraction. TMADH is a complex iron-sulfur flavoprotein of 147,000 daltons composed of two non-identical subunits of about 75,000 daltons each. It contains one Fe4S4 cluster and one covalently bound FMN coenzyme per molecule. The enzyme catalyzes the oxidation of trimethylamine to dimethylamine and formaldehyde with subsequent electron transfer to the artificial acceptor phenazine methosulfate. When reduced by substrate the enzyme exhibits very strong spin coupling between the reduced cofactors accompanied by an apparent conformation change. Crystals of TMADH are monoclinic, space group P21 with cell dimensions a=147.63A, b=71.96A, c=83.66A and Beta=97.64A. The crystals diffract to 2.0A resolution. The structure will be analyzed by the isomorphous replacement method first at 6.0A and then at 2.4A resolutions. The 6.0A map will be studied to identify the domain and subunit structure of the enzyme. Native anomalous scattering data will be analyzed to locate the iron-sulful cluster. The 2.4A map will be used to trace the polypeptide chain and locate the orientation of the side chains and of the cofactors. A model of the protein will be constructed on the MMS-X molecular graphics system. In order to study the enzymatic mechanism, various substrates, inhibitors and allosteric effectors will be bound to the enzyme and analyzed by difference Fourier methods. Finally, the structure will be refined to 2.0A resolution by the Hendrickson-Konnert restrained least squares procedure.

三甲基胺脱氢酶（TMADH）的分子结构 甲基营养菌W3 A1的X射线衍射分析。 TMADH 是一种147，000道尔顿的复杂铁硫黄素蛋白，由两个 每个约75，000道尔顿的不相同亚基。 它含有一个Fe 4S 4 簇和每个分子一个共价结合的FMN辅酶。 酶 催化氧化三甲胺为二甲胺和甲醛 随后电子转移到人工受体吩嗪 甲硫酸盐 当被底物还原时，该酶表现出非常强的 约化辅因子之间的自旋耦合伴随着明显的 构象变化 TMADH晶体为单斜晶系，空间群P21 晶胞尺寸a= 147.63 A，B= 71.96 A，c= 83.66 A和β = 97.64 A。 的 晶体的分辨率为2.0A。 该结构将通过以下方式进行分析： 同晶置换法先在6.0A后在2.4A 决议。 将研究6.0A图以确定域， 酶的亚基结构。 天然异常散射数据将是 分析以定位铁硫簇。 2.4A地图将用于 追踪多肽链并定位侧链的方向 和辅因子的关系。 蛋白质的模型将在 MMS-X分子图形系统。 为了研究 机制，各种底物，抑制剂和变构效应物将被 与酶结合并通过差分傅立叶方法分析。 最后， 结构将由Hendrickson-Konnert改进到2.0A分辨率 约束最小二乘法