How enzymes break carbon-fluorine bonds

酶如何打破碳氟键

• 批准号: 170109-2010
• 负责人: Pai, Emil
• 金额: $ 6.07万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2012
• 资助国家: 加拿大
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=513862
• 关键词: How; enzymes; break; carbon; fluorine

Dehalogenase enzymes break carbon-halogen bonds. Most remarkable are enzymes able to break the most stable bond in organic chemistry, the carbon-fluorine bond. Supported by an NSERC Strategic Grant and in collaboration with E. Edwards (U of T) and A.Yakunin (SGC), we screened microbial genomes for dehalogenases. We raised the number of confirmed dehalogenases from 2 to 20, 9 of them defluorinases and 4 representing the first defluorinase members of the L-2-haloacid dehalogenase protein family. We will determine the molecular interactions that form the basis of their catalytic power, contributing to biodegradation solutions for most recalcitrant environmental pollutants. We have used ITC to determine kinetic parameters of native and mutant enzymes. The fluoroacetate dehalogenase RPA1163 from R. palustris was characterized in detail. Its static high-resolution crystal structures (~ 1.2 Å; native and mutants) with various ligands provided a set of 'snapshots' of the catalytic intermediates. They also indicated transient binding sites as the substrate approaches the catalytic machinery and rotational movements upon transformation. Our static analyses showed that the crystals accommodate the complete catalytic cycle without compromising diffraction power. We have synthesized the "caged" 1-(2-nitrophenyl) ethyl-derivative of the substrate. Short UV-laser pulses can transform this "caged compound" into the substrate, initiating its binding and catalytic conversion. Applying time- resolved (TR)-Laue techniques, we will collect diffraction data as a function of time since initiation and convert them to a "movie" of the catalytic reaction, allowing observation in almost atomic detail. We collaborate with the world's foremost experts in laser-triggered TR-Laue-diffraction to accelerate our progress and secure timely access to their superb resources. Our system will also serve as a test case for developing monochromatic methods for TR crystallography. In addition, we plan to engineer a light-sensitive dehalogenase by fusing a light-trigger LUV domain to the defluorinase, which will allow us to preform the substrate complex, thereby shifting the reaction from a bimolecular to a unimolecular one, allowing for easier data interpretation.

脱氧酶酶破坏碳纤维键。最引人注目的是能够打破有机化学中最稳定键的酶，即碳氟键。在NSERC的战略赠款的支持下，并与E. Edwards（U的U of T）和A.yakunin（SGC）合作，我们为脱核酶筛选了微生物基因组。我们将确认的脱酰酶的数量从2个增加到20，其中9个是脱氟辛酶，4个代表L-2-半酸脱核酶蛋白蛋白家族的第一个放流酶成员。我们将确定构成其催化能力基础的分子相互作用，这有助于大多数顽固环境污染物的生物降解溶液。我们已经使用ITC来确定天然和突变酶的动力学参数。详细介绍了来自R. palustris的氟乙酸脱核酶RPA1163。它的静态高分辨率晶体结构（〜1.2Å；天然和突变体）具有各种配体，提供了一组催化中间体的“快照”。他们还指出，底物在转化时接近催化机制和旋转运动时表示瞬态结合位点。我们的静态分析表明，晶体可容纳完整的催化循环，而不会损害衍射功率。我们已经合成了底物的“笼” 1-（2-硝基苯基）乙基衍生物。短的紫外线脉冲可以将这种“笼化合物”转化为底物，启动其结合和催化转化。应用时间解析（TR）-Laue技术，我们将收集衍射数据作为时间的函数，因为开始并将其转换为催化反应的“电影”，从而几乎可以进行原子细节观察。我们与世界上最重要的专家在激光触发的TR-Laue-Diffraction方面合作，以加速我们的进度并及时访问其出色的资源。我们的系统还将作为开发单色方法用于TR晶体学的测试案例。此外，我们计划通过将光触发的LUV结构域融合到放流酶来设计光敏的脱核酶，这将使我们能够预先形成底物络合物，从而将反应从双分子转移到非分子的反应，从而更容易地解释数据。