CAS: Enzymatic Hydrolytic Dehalogenation of Chlorinated Aromatic Compounds

获得该奖项的是科罗拉多矿业学院生命过程化学系的Holz博士，他研究了百菌清脱卤酶(CHD)如何在环境条件下选择性地将一个芳香碳氯(C-Cl)键转化为百菌清(TPN；2，4，5，6-四氯间苯二甲腈)上的一个芳香碳氢氧化物(C-OH)键。TPN是一种在美国广泛使用的杀菌剂；在美国，每年有500多万公斤TPN喷洒在水果和农作物上。TPN在土壤中被强烈吸收，特别是具有高有机质的土壤，如在水环境中发现的土壤，在那里它可以保留并污染地下水多年。TPN对鱼类和其他水生物种以及鸟类和无脊椎动物都有剧毒。TPN也是对人体皮肤和眼睛的刺激物，可导致严重的胃肠道问题，已被美国环境保护局(EPA)列为可能的人类致癌物质。CHD是TPN生物修复过程中的一种重要酶，因此越来越被认为是一种新的“绿色”生物催化剂。霍尔茨博士的研究将使用跨学科的实验方法，深入了解冠心病是如何分解TPN的。了解CHD的催化机制将有助于深入了解其生物修复潜力，并有可能通过仿生和蛋白质工程方法促进这种催化化学的扩展。这些研究活动为实施这项工作的本科生和研究生创造了一个激励的智力环境。霍尔茨博士还侧重于(I)通过招生扩大STEM职业中代表性不足背景的学生的参与，以及(Ii)增加本科生对研究的参与。卤代芳香族化合物具有较高的毒性和致癌性，是公认的主要有机污染物。这些化合物以工业过程中产生的污染物的形式进入环境，在工业过程中它们被广泛用作溶剂、脱脂剂和杀菌剂。由于这些化合物的化学稳定性、耐降解性和亲脂性，从环境中去除这些化合物是具有挑战性的。将百菌清脱卤酶等酶应用于卤代芳香族化合物污染环境的生物修复，需要在分子水平上了解CHD催化TPN降解的机理。该奖项支持的研究将集中于回答关于CHD催化作用的三个具体问题：(I)卤代芳香反应底物对CHD的识别和结合的决定因素是什么？(Ii)底物是否与酶活性部位的锌离子结合，过渡态的结构是什么？以及(Iii)CHD活性部位的哪些残基对催化作用是重要的？霍尔茨博士和他的团队将结合使用蛋白质生物化学、光谱学和酶动力学分析、计算化学和X射线结晶学研究。该项目的成功完成将加深我们对CHD催化机理的了解，并促进用于合成、生物催化和生物修复的工程化催化剂的开发。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes in the Chemistry Division is funding Dr. Holz from the Colorado School of Mines to investigate how the enzyme chlorothalonil dehalogenase (Chd) selectively converts under ambient conditions an aromatic carbon-chlorine (C-Cl) bond to an aromatic carbon-hydroxide (C-OH) bond on chlorothalonil (TPN; 2,4,5,6-tetrachloroisophthalonitrile). TPN is a fungicide that is widely used in the US; more than five million kilograms of TPN are sprayed every year on fruits and crops in the US. TPN is strongly absorbed in soil, particularly soil with high organic matter such as that found in aquatic environments, where it can remain and contaminate groundwater for years. TPN is highly toxic to fish and other aquatic species as well as to birds and invertebrates. TPN is also an irritant for human skin and eyes that can cause severe gastrointestinal issues and has been classified by the U.S. Environmental Protection Agency (EPA) as a probable human carcinogen. Chd is an important enzyme in the bioremediation of TPN and is thus becoming increasingly recognized as a new “Green” biocatalyst. Dr. Holz’s research will use an interdisciplinary experimental approach to gain insight into how TPN is hydrolyzed by Chd. Understanding the catalytic mechanism of Chd would provide insight into its bioremediation potential and potentially facilitate the expansion of this catalytic chemistry through biomimetic and protein engineering approaches. These research activities create a stimulating intellectual environment for the undergraduate and graduate students who implement the work. Dr. Holz also focuses on (i) broadening participation of students from backgrounds underrepresented in STEM careers via student recruitment, and (ii) increase undergraduate participation in research. Halogenated aromatic compounds are known to be major organic pollutants given their high toxicity and carcinogenic properties. These compounds enter the environment as contaminants derived from industrial processes where they are widely used as solvents, defatting agents, and fungicides. The removal of these compounds from the environment is challenging because of their chemical stability, resistance to degradation, and lipophilic properties. Use of an enzyme such as chlorothalonil dehalogenase in the bioremediation of environments contaminated with halogenated aromatic compounds is conditioned upon the understanding at molecular level of the mechanism of the Chd-catalyzed hydrolysis of TPN. The research supported by this award will focus on answers three specific questions about the Chd catalytic role: (i) What are the determinants of the recognition and binding of the halogenated aromatic reaction substrates to Chd?, (ii) Does substrate bind to the Zn(II) ions in the active site of the enzyme and what is the structure of the transition state?, and (iii) Which residues in the active site of Chd are important for the catalysis? Dr. Holz and his team will use a combination of protein biochemistry, spectroscopy and enzyme kinetic analysis, computational chemistry, and X-ray crystallographic studies. The successful completion of this project will enhance our understanding of the catalytic mechanism of Chd and stimulate development of engineered catalysts for deployment in synthesis, biocatalysis and bioremediation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.