Chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) is a fungicide, probable human carcinogen, and major environmental toxin. This compound also has low solubility in water but can bioaccumulate in soil and aquatic environments where it can be stable for more than one year. Hydrolytic dehalogenation is a promising bioremediation tool to degrade TPN into the less toxic 4-hydroxytrichloro-isophthalonitrile (4-OH-TPN) product that does not bioaccumulate. The chlorothalonil dehalogenase from <i>Pseudomonas sp. </i>CTN-3 (Chd) is a Zn(II)-dependent hydrolytic dehalogenase with selective activity towards TPN. Our work is motivated by the lack of understanding of Chd’s catalytic mechanism. It is a 36 kDa protein with an αββα-sandwich fold that has one structural Zn(II) at the interface of the dimers and one catalytic Zn(II) ion in the active site pocket. This catalytic Zn(II) is a trigonal bipyramidal coordinated by His117, His257, Asp116, Asn216, and a water/hydroxide (Figure 1). This water/hydroxyl is stabilized by an hydrogen bond from His114. Based on the x-ray structure, His114 has two distinct spatial conformations, shown in figure 1. EPR data on the catalytically active Co(II)-substituted form of Chd confirmed that there are two likely His114 conformations. EPR data obtained on substrate analog inhibitors provided hyperfine split signals similar to the un-complexed enzyme, suggesting that substrate analog inhibitors do not bind directly to the catalytic metal center. EPR spectra of phosphonic and boronic acids, which are possible transition-state analogs, were also obtained and suggest binding directly to the metal center. These data are consistent with the previously proposed catalytic mechanism for Chd, which suggested direct binding of TPN to the Chd active site Zn(II) ion.

EPR of the hydrolytic dehalogenase from <i>Pseudomanas sp</i>. CTN-3

ACS Spring 2022

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EPR of the hydrolytic dehalogenase from Pseudomanas sp. CTN-3