Allostery-Driven Substrate Gating in the Chlorothalonil Dehalogenase from Pseudomonas sp. CTN-3

The catalytic mechanism of the Zn(II)-dependent chlorothalonil dehalogenase from <i>Pseudomonas</i> sp. CTN-3 (Chd) was examined using molecular dynamics (MD) simulations, Bayesian network analysis, and Markov state model analysis to quantify its motions. Chd selectively substitutes an aromatic chlorine-carbon bond in chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) with an aromatic alcohol (4-hydroxytrichloro-isophthalonitrile; 4-OH-TPN). It is a homodimer with two solvent-accessible channels in each monomer, which are proposed to provide different routes for substrate and products to access/leave the catalytic Zn(II) site. Based on MD simulations, Chd exhibits allosteric behavior wherein a "Y"-shaped substrate channel exhibits a "flip flop" mechanism, where the "right" substrate channel opens to allow TPN to enter, after which it closes, followed by the "left" channel opening. The "right" channel then reopens, likely to allow the product, 4-OH-TPN, to leave the active site, but this reopening of the right channel drives the "left" channel to close. Coupled with the substrate channels alternately opening and closing, a corresponding possible Cl^- channel opens and closes. Although the dynamics of this process are fast, Chd needs to overcome a 5 kT free-energy barrier for this transition and to relax after opening. Additionally, exposed "wing" residues, hydrophilic residues at the ends of protruding α-helices, act as allosteric indicators, signaling the complex allosteric motions required to open the substrate channel. We propose, for the first time, a dynamic mechanism that drives substrate binding and product release, providing new insight into Chd's catalytic mechanism.