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

## TL;DR

This paper explores how a bacterial enzyme uses allostery to control substrate entry and product exit, revealing a dynamic 'flip flop' mechanism in its catalytic process.

## Contribution

The study proposes a novel dynamic mechanism for substrate binding and product release in Chd, driven by allostery and molecular motions.

## Key findings

- Chd exhibits a 'flip flop' mechanism where one substrate channel opens while the other closes.
- Allosteric behavior in Chd is linked to molecular motions and a free-energy barrier of 5 kT.
- Exposed 'wing' residues act as allosteric indicators for channel opening.

## Abstract

A structural dynamics study of Chd using molecular dynamics simulations, Bayesian network analysis, and Markov state model analysis to quantify observed motions. Chd exhibits allosteric behavior wherein a “Y”-shaped substrate channel exhibits a “flip flop” mechanism, opening only one side of a dimer to substrate at any time. These data link allostery between monomers and a stable but highly dynamic protein that leverages molecular motions to enable catalysis, providing new insight into the catalytic mechanism of Chd.

The catalytic mechanism of the Zn(II)-dependent chlorothalonil dehalogenase from Pseudomonas 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.