Anoxic Enzymatic Conversion of Acetylene

乙炔的缺氧酶促转化

• 批准号: 210678598
• 负责人: Professor Dr. Oliver Einsle
• 金额: --
• 依托单位: Institut für Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/210678598?language=en
• 关键词: Anoxic; Enzymatic; Conversion; Acetylene

The gas acetylene (ethyne) undergoes two known enzymatic conversions in nature. Nitrogenase, the enzyme required for the reductive fixation of dinitrogen into bioavailable ammonium, reduces acetylene in a two-electron step to ethylene (ethene). In contrast, the tungsten / [4Fe:4S] enzyme acetylene hydratase catalyzes the hydratation of the gas to yield acetaldehyde. The proteins are well characterized structurally and functionally, but the mode and exact site of acetylene binding and the mechanism of its conversion remain under heavy debate for both cases. Clearly, the mechanisms of acetylene conversion follow entirely different routes: while nitrogenase most likely binds acetylene (and other substrates) to the [Mo:7Fe:9S:X]:homocitrate FeMo cofactor, from which electrons of a low redox potential get transferred directly to the substrate, the tungstoprotein acetylene hydratase has a distinct, binding pocket pre-formed by a ring of hydrophobic amino acids. Here the reaction may not involve the formation of a metal-carbon bond, but rather the activation of a water molecule coordinated to tungsten that subsequently attacks the triple bond of the alkyne. We will establish and optimize both native and recombinant production of acetylene hydratase in our laboratory, create point-specific mutants that will be analyzed for enzymatic activity and obtain high-resolution structures of wild type and variant proteins pressurized with acetylene or other candidate ligands. Nitrogenase will be isolated from Azotobacter vinelandii and assayed for acetylene reduction activity. Acetylene, and in particular the strong ligand CO will be used in pressurization experiments to obtain atomic resolution data from which we expect to gain essential mechanistic clues.

气体乙炔（乙炔）在自然界中经历两种已知的酶促转化。固氮酶是将二氮还原固定为生物可利用的铵所需的酶，它在两个电子步骤中将乙炔还原为乙烯。相比之下，钨/ [4Fe：4S]酶乙炔水合酶催化气体的水合以产生乙醛。这些蛋白质在结构和功能上都有很好的特点，但乙炔结合的模式和确切位点及其转化机制仍然存在很大的争议。显然，乙炔转化的机制遵循完全不同的路线：固氮酶最有可能将乙炔（和其他底物）结合到[Mo：7 Fe：9 S：X]：高柠檬酸FeMo辅因子，低氧化还原电位的电子直接转移到底物，而钨蛋白乙炔水合酶具有由疏水氨基酸环预先形成的独特结合口袋。在这里，反应可能不涉及金属-碳键的形成，而是与钨配位的水分子的活化，其随后攻击炔的三键。我们将在我们的实验室建立和优化乙炔水合酶的天然和重组生产，创建点特异性突变体，这些突变体将用于分析酶活性，并获得用乙炔或其他候选配体加压的野生型和变体蛋白质的高分辨率结构。将从棕色固氮菌中分离固氮酶，并测定其乙炔还原活性。乙炔，特别是强配体CO将用于加压实验，以获得原子分辨率的数据，我们希望从中获得必要的机制线索。