CAS: Enzymatic Hydrolytic Dehalogenation of Chlorinated Aromatic Compounds

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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.