How enzymes break carbon-fluorine bonds

脱卤酶破坏碳-卤素键。最引人注目的是能够破坏有机化学中最稳定的键——碳氟键的酶。在NSERC战略基金的支持下，我们与E. Edwards （university of T）和A.Yakunin （SGC）合作，筛选微生物基因组中的脱卤酶。我们将确认的去卤酶的数量从2个增加到20个，其中9个是去氟酶，4个是l -2-卤酸去卤酶蛋白家族的第一个去氟酶成员。我们将确定形成其催化能力基础的分子相互作用，为大多数顽固的环境污染物提供生物降解解决方案。我们用ITC测定了原生酶和突变酶的动力学参数。对帕氏鼠氟乙酸脱卤酶RPA1163进行了详细的鉴定。它的静态高分辨率晶体结构（~ 1.2 Å；原生和突变体）与各种配体提供了一组催化中间体的“快照”。他们还指出了底物接近催化机制时的瞬时结合位点和转化时的旋转运动。我们的静态分析表明，晶体适应完整的催化循环而不影响衍射功率。我们合成了底物的“笼状”1-（2-硝基苯基）乙基衍生物。短紫外激光脉冲可以将这种“笼状化合物”转化为衬底，启动其结合和催化转化。应用时间分辨(TR)-劳埃技术，我们将收集衍射数据作为起始时间的函数，并将其转换为催化反应的“电影”，允许几乎原子细节的观察。我们与世界上最重要的激光触发tr -劳厄衍射专家合作，以加快我们的进展，并确保及时获得他们的优质资源。我们的系统也将作为开发单色方法的测试用例。此外，我们计划通过将光触发LUV结构域融合到脱氟酶上来设计一种光敏脱卤酶，这将使我们能够预先形成底物复合物，从而将反应从双分子转变为单分子，从而使数据解释更容易。

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.