The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation

Abstract

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole-enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen-bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the alpha-carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C-F bond is greatly facilitated by the hydrogen-bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…49) To build an initial structure for the QM/MM calculations, the binding mode of fluoroacetate in the active site of fluoroacetate dehalogenase was estimated from the crystal structure of haloalkane dehalogenase complexed with 1,2-dichloroethane. The chlorine atom hydrogen-bonded to the halide ion acceptors (Trp125 and Trp175) of haloalkane dehalogenase was replaced by a fluorine atom, and the other chlorine atom was replaced by a carboxyl oxygen atom.…”

Biodegradation and biotransformation of organofluorine compounds

“…Site directed mutagenesis studies (Trp105Ala) revealed that this residue is critical for activity towards fluoroacetate, but not chloroacetate, and it was proposed that hydrogen bonding between fluorine and Trp150 is a key factor in reducing the activation energy required to cleave the C-F bond. This suggestion is supported by quantum mechanical/molecular mechanical (QM/ MM) calculations, which identified Trp150, His149 and Tyr212 as residues that form hydrogen bonds with fluorine to promote C-F cleavage and stabilise the fluoride ion (Kamachi et al 2009). The fluoroacetate-degrading bacterium Pseudomonas fluorescens DSM 8341 is a convenient model to study the characteristics of organofluorine catabolism in biofilm reactors.…”

Theoretical prediction and experimental verification on enantioselectivity of haloacid dehalogenase l-DEX YL with chloropropionate

“…Site-directed mutagenesis experiments have revealed that some amino acid residues are essential for the catalytic function of l-DEX YL [6]. 18 Oincorporation experiments have revealed that Asp10 plays a pivotal role in a nucleophilic attack on a substrate ␣-carbon to form an ester intermediate [13]. These studies suggest the reaction mechanism shown in Figure 1.…”

“…These experimental results indicate that the catalytic reaction was prevented at least in one of the two processes, substrate binding process and dehalogenation reaction process. In addition to haloacid dehalogenase, the TS structures in haloalkane dehalogenase [16,17] and in fluoroacetate dehalogenase [18] have been computationally determined to elucidate their catalytic mechanisms. A couple of theoretical studies on the mechanism of the specific substrate recognition in dehalogenases have been reported [19][20][21][22].…”

“…strain YL cell culture in a medium containing 2-chloropropionate (2-CPA) as the sole carbon source [5]. The reaction mechanism of l-DEX YL has been analyzed by site-directed mutagenesis [6], X-ray crystallography [7][8][9], mass spectrometry [10][11][12], 18 O-incorporation experiments [13], and computer simulations with molecular dynamics (MD), fragment molecular orbital (FMO) [14] and quantum-mechanical/molecularmechanical (QM/MM) methods [15]. Site-directed mutagenesis experiments have revealed that some amino acid residues are essential for the catalytic function of l-DEX YL [6].…”