AUTHOR=Yang Xinhang , Diviesti Karla , Miller Callie , Bennett Brian , Holz Richard C. 

TITLE=Insights into the catalytic mechanism of the chlorothalonil dehalogenase from Pseudomonas sp. CTN-3

JOURNAL=Frontiers in Chemical Biology

VOLUME=2

YEAR=2023

URL=https://www.frontiersin.org/journals/chemical-biology/articles/10.3389/fchbi.2023.1105607

DOI=10.3389/fchbi.2023.1105607

ISSN=2813-530X

ABSTRACT=<p>The catalytically competent Co(II)-loaded form of the chlorothalonil dehalogenase from <italic>Pseudomonas</italic> sp. CTN-3 (Chd, EC 3.8.1.2) was characterized by kinetic and spectroscopic methods. Maximum chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) dehalogenase activity was observed in the presence of one Co(II) ion per monomer with <italic>k</italic><sub>cat</sub> and <italic>K</italic><sub>m</sub> values of 12 ± 3 s<sup>−1</sup> and 130 ± 10 μM, respectively, providing a catalytic efficiency (<italic>k</italic><sub>cat</sub>/<italic>K</italic><sub>m</sub>) of ∼9.2 × 10<sup>4</sup> M<sup>−1</sup>s<sup>−1</sup>. The dissociation constant (<italic>K</italic><sub>d</sub>) for Co(II) was determined to be 0.29 µM, and UV-Vis spectroscopy indicated the active site Co(II) ion resides in a penta-coordinate environment. EPR spectra of Co<sub>1</sub>-Chd contain at least three distinct signals, an <italic>M</italic><sub><italic>S</italic></sub> = ± <sup>1</sup>/<sub>2</sub> signal with a ∼94 G <sup>59</sup>Co hyperfine pattern centered at <italic>g</italic><sub>1</sub>’ ≅ 6.7, a broader <italic>M</italic><sub><italic>S</italic></sub> = ± <sup>1</sup>/<sub>2</sub> signal with <italic>g</italic><sub>1</sub>’ ≅ 5.7, an <italic>M</italic><sub><italic>S</italic></sub> = ± <sup>3</sup>/<sub>2</sub> signal with tentatively estimated parameters of <italic>g</italic><sub>1</sub>’ ≅ 10.5 (<italic>g</italic><sub><italic>z</italic></sub> = 2.75), <italic>A</italic><sub>1</sub>(<sup>59</sup>Co) ≅ 110 G, and a high-field broad resonance at <italic>g</italic><sub>3</sub>’ ≅ 1.8. Four substrate-analog inhibitors with IC<sub>50</sub> values ranging from 110 μM to 19 mM were also identified and characterized. Upon the addition of each of the substrate-like inhibitors to Co<sub>1</sub>-Chd, changes in the EPR spectrum were observed that, in all cases, were simpler than that of Co<sub>1</sub>-Chd in the absence of inhibitors and could be simulated as either a single species or a mixture of two. Simulation of these data indicate that the corresponding EPR signals are each due to a ground state <italic>M</italic><sub><italic>S</italic></sub> = <sup>1</sup>/<sub>2</sub> Kramers’ doublet and are consistent with pentacoordinate Co(II) with a relatively constrained coordination sphere. These data suggest that the nitrile moiety of TPN may not directly coordinate to the active site metal ion, providing new insight into the catalytic mechanism for Chd.</p>