Theoretical Study On Catalytic Mechanism Of Dehalogenation Catalyzed By Dehalogenases

Dehalogenases comprise such a group of microbial enzymes that catalyze the cleavage of carbon–halogen bonds into the corresponding alcohols and halide anions. There is a growing interest in the application of these enzymes as industrial biocatalysts. Recently, the catalytic prosperities of these enzymes in bioremediation applications have therefore been subjected to detailed investigations.To date, there are five bacterial dehalogenases, including haloalkane dehalogenases, haloacid dehalogenases, 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase, haloalcohol dehalogenase and a trans-3-chloroacrylic acid dehalogenase. These enzymes make use of a variety of distinctly different catalytic mechanisms to cleave carbon–halogen bonds. It is demonstrated the power of substitution mechanisms of the first three enzymes that proceed via a covalent aspartyl intermediate. For the latter two homologous ones, it is exploited that their catalytic mechanisms are different in different chemical environments. Although the five dehalogenases are very similar, they have some difference in the geometry and size of the active site cavity and differences in the way in which the leaving group is stabilized. Comparison of them is important for explaining the molecular evolution and enhancing the enzyme catalysis by mutations. There are so many kinds of bacterial dehalogenases that together they cover a broad range of substrates, halogenated compounds.Haloalkane dehalogenases (EC 3.8.1.5) are a typical and widely-used bacterial dehalogenases. The mechanism of haloalkane dehalogenases became apparent when the structure of the Xanthobacter autotrophicus enzyme (DhlA) was solved by X-ray crystallography. Many studies have demonstrated that the enzymatic hydrolysis follows a two-step process with the formation of a covalent alkyl-enzyme ester intermediate. However, mechanistic issues about the first SN2 nucleophilic displacement reaction and the ester-enzyme hydrolysis reaction are still uncertain. In this paper, the first SN2 nucleophilic displacement reactions catalyzed by enzyme DhlA and LinB and the ester-enzyme hydrolysis reaction were investigated in detail by using density functional theory to explain the origin of enzyme catalysis. The mechanism investigations play an important role in explaining the origin of enzyme catalysis aimed at improving the enzyme activity and enhancing the substrate specificity.The most important results are as follows:(1) The SN2 nucleophilic displacement reaction catalyzed by enzyme DhlA was investigated in detail by using density functional theory. In enzyme DhlA catalysis, the carbon atom attached to the leaving halogen of the substrate is attacked by the carboxylate group of Asp124 in the SN2 fashion, yielding an alkyl-enzyme intermediate and a chlorine ion. Different hydrogen bonds patterns of the oxyanion hole and the halide-stabilizing residues, Trp125 and Trp175 play an important role in the dehalogenation reaction. The hydrogen bonds of the oxyanion hole make the carboxylate oxygen of Asp124 closer to DCE so that the reacting fragment is more reactive. Trp125 has a major stabilization effect on the reactant complex. The oxyanion hole and the halide-stabilizing residues concertedly cause an earlier TS with the activation barrier of 16.60 kcal/mol. The stabilization effect of Trp125 and Trp175 on Cl1 atom in TS1 is larger than that of RC1 by 15.67 kcal/mol so that they make contribution to the stabilization of the transition state. Moreover, analysis of the solvent effect on the reaction shows that enzyme catalysis is due to reactant-state destabilization relative to water solution. So the enzymatic action can be attributed to a combination of reactant-state destabilization and TS electrostatic stabilization. They lower the energy barrier by 6.12 kcal/mol.DhlA and LinB are homologous enzymes. Analysis of the activation barriers reveals that the reaction process catalyzed by LinB is very similar to that of DhlA. However, their catalytic triad and active sites are different. DCE is able to benefit from substantial conformational freedom within the active site. The preponderant conformer is important for the reaction to proceed. For DhlA catalysis, the gauche conformer (D (Cl1–C1–C2–Cl2) = -60°) of the substrate is easier to take place, whereas the same conformer of the substrate is difficult to dehalogenate in LinB.(2) The ester-enzyme hydrolysis reaction catalyzed by enzyme DhlA was investigated in detail using density functional theory. The ester-enzyme hydrolysis reaction involves a nucleophilic addition of a water molecule to the carbonyl group, which is catalyzed by the general base His289 and is accompanied by a proton transfer from the nucleophilic water. The ester is hydrolyzed via a TI and it splits to a corresponding alcohol and acid (Asp124). After TI, elimination and another proton transfer process take place. Herein whether the transferred proton is from protonated His289 or deprotonated water nucleophile was elucidated. In both of the two pathways, the first AdN step is rate-limiting. In the gas phase, the barrier for the pathway with the transferred proton from protonated His289 is lower than that of the pathway with the transferred proton from deprotonated water molecular by 0.13 kcal/mol. Thus the former is the preponderant process. In benzene, the barrier for the dominant pathway is lower than that of the inferior pathway by 0.67 kcal/mol.When a second water molecule involved in the X-ray structure of DhlA was also selected, the hydrolysis mechanism is different. Unlike three steps in the pathway with the transferred proton from protonated His289, only two steps were involved in the water assisted hydrolysis. The first AdN step is followed by the concerted step of elimination and the second proton transfer. The overall reaction is also dominated by the first AdN step. In the gas phase, the hydrolysis can be accelerated by lowering the barrier, 3.35 kcal/mol. Moreover, benzene can make an effect on promoting ester hydrolysis, lowering the barrier by 1.79 kcal/mol. The obtained activation barrier in benzene, 18.65 kcal/mol is in fairly good agreement with the recently reported barrier of the AdN step, 19.5±2.1 kcal /mol. Therefore both the second water molecule and the protein environment play an important role in hydrolytic dehalogenation.