Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation.

Panu Pimviriyakul,Pimchai Chaiyen,Panida Surawatanawong

doi:10.1039/c8sc01482e

Abstract

Enzymes that are capable of detoxifying halogenated phenols (HPs) and nitrophenols (NPs) are valuable for bioremediation and waste biorefining. HadA monooxygenase was found to perform dual functions of oxidative dehalogenation (hydroxylation plus halide elimination) and denitration (hydroxylation plus nitro elimination). Rate constants associated with individual steps of HadA reactions with phenol, halogenated phenols and nitrophenols were measured using combined transient kinetic approaches of stopped-flow absorbance/fluorescence and rapid-quench flow techniques. Density functional theory was used to calculate the thermodynamic and electronic parameters associated with hydroxylation and group elimination steps. These parameters were correlated with the rate constants of hydroxylation, group elimination, and overall product formation to identify factors controlling individual steps. The results indicated that the hydroxylation rate constant is higher when the pK a of the phenolic group is lower, i.e. it is more easily deprotonated, but not higher when the energy gap between the E LUMO of the C4a-hydroperoxy-FAD intermediate and the E HOMO of the phenolate substrate is lower. These data suggest that the substrate deprotonation has a higher energy barrier than the -OH transfer, and thus controls the hydroxylation step. For the group elimination, the process is controlled by the ability of the C-X bond to break. For the overall product formation (hydroxylation and group elimination combined), this analysis showed that the rate constant of product formation is dependent on the pK a value of the substrate, indicating that the overall reaction is controlled by substrate deprotonation. This step also likely has the highest energy barrier and thus controls the overall process of oxidative dehalogenation and denitration by HadA. This report is the first to identify a key mechanistic factor controlling the enzymatic processes of oxidative dehalogenation and denitration.

Highlights

Halogenated phenols (HPs) and nitrophenols (NPs) such as 4-chlorophenol (4-CP), 4-nitrophenol (4-NP), 3,5-dibromo-4hydroxybenzonitrile, chloronitroaromatic compounds, 2,4,6trichlorophenol (2,4,6-TCP), and 2,4,5-trichlorophenol (2,4,5TCP) are among the top agrochemicals widely used around the world as pesticides, disinfectant agents, wood preservatives and dyes.[1,2,3] Due to their heavy usage and resistance to degradation, these compounds have been found to accumulate widely in soil and water.[1,4,5] This creates serious concerns for human health, as these compounds are assimilated into the food chainA variety of technologies for degrading halogenated phenols (HPs) and NPs have been developed
Previous studies have shown that hydroxy-FAD intermediate (HadA) can use CPs (4-CP, 2-CP, 2,4-DCP, 2,4,5-TCP, 2,4,6-TCP and 2,5-DCHQ) as substrates[31,33] but it was not known whether the enzyme can dehalogenate other HPs
In order to explore whether HadA can use HPs and NPs as substrates, multiple turnover reactions of HadA with HPs and NPs were performed, and the reactions were monitored by absorption spectroscopy

Summary

Introduction

Halogenated phenols (HPs) and nitrophenols (NPs) such as 4-chlorophenol (4-CP), 4-nitrophenol (4-NP), 3,5-dibromo-4hydroxybenzonitrile, chloronitroaromatic compounds, 2,4,6trichlorophenol (2,4,6-TCP), and 2,4,5-trichlorophenol (2,4,5TCP) are among the top agrochemicals widely used around the world as pesticides, disinfectant agents, wood preservatives and dyes.[1,2,3] Due to their heavy usage and resistance to degradation, these compounds have been found to accumulate widely in soil and water.[1,4,5] This creates serious concerns for human health, as these compounds are assimilated into the food chain. A variety of technologies for degrading HPs and NPs have been developed. These include physical, chemical and biological processes. Incineration of chlorophenol (CP) wastes is known to produce compounds with even greater toxicity, such as polychlorinated dibenzo-p-dioxins or polychlorinated dibenzofurans.[11,12,13] On the other hand, biological degradation of HPs can be performed through efficient and clean processes.

Results

Discussion

Conclusion