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

Pimviriyakul, Panu; Surawatanawong, Panida; Chaiyen, Pimchai

doi:10.1039/c8sc01482e

Cited by 37 publications

(58 citation statements)

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“…Based on all data, the enzymatic had system for HPs/NPs degradation by the three flavin-dependent enzymes, HadA monooxygenase coupled with HadX flavin reductase and HadB quinone reductase, can be summarized as in Fig. 12 (16,17). Understanding the complete metabolic pathway for the biodegradation of toxic aromatic compounds is useful for future development and optimization to increase the efficiency of the system.…”

Section: Catalytic Mechanisms Of Hadx Reductase and Hadb Reductase Thmentioning

confidence: 99%

“…Several aerobic bacteria are capable of converting HPs or NPs to compounds that are ready to be assimilated via central metabolic pathways (5, 6, 8 -15). Among these bacteria, the had operon from Ralstonia pickettii DTP0602 is particularly interesting because one of the key enzymes that catalyze a crucial step in detoxification, HadA monooxygenase, has recently been reported to have dual activities in catalyzing hydroxylation and group elimination of HPs (dehalogenation) and NPs (denitration) to generate a quinone product (16). The resulting quinone needs to be further reduced to hydroquinone, which can be further metabolized by other enzymes in the had operon as shown in Fig.…”

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confidence: 99%

“…1. In previous experiments, ascorbic acid was used as a reductant to generate hydroquinone products in vitro (16,17). It is still unknown how quinone is converted into hydroquinone in vivo.…”

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confidence: 99%

“…strates in the reaction is the key step that controls the overall reaction of hydroxylation and the group elimination steps (16,17). As HadA belongs to the class of two-component flavin-dependent monooxygenases, a flavin reductase is required to supply reduced flavin to the system (18).…”

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confidence: 99%

“…The resulting reduced flavin generally does not bind tightly to the reductase, and thus dissociates from the enzyme to bind to and act as a substrate for the monooxygenase (18 -20). Previously, the reductase component of the p-hydroxyphenylacetate hydroxylase system (C 1 ) (19,20) was used as a flavin reductase to supply FADH Ϫ for HadA (16,17). The partner reductase that was co-evolved with HadA for catalyzing dehalogenation and denitration with HadA is currently unknown (Fig.…”

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confidence: 99%

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A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

Pimviriyakul

Chaiyen

2018

Journal of Biological Chemistry

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Edited by F. Peter GuengerichHalogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the had operon from the bacterium Ralstonia pickettii DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH ؊ )-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH ؊ for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH ؊ for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH؊ and that HadX can transfer FADH ؊ from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH ؊ for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, i.e. HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications. 2 The abbreviations used are: HPs, halogenated phenol; HadA, two-component FADH Ϫ utilizing monooxygenase; HadB, a quinone reductase encoded in the had operon; HadX, a flavin reductase encoded in the had operon; NPs, nitrophenols; FADH Ϫ , reduced flavin adenine dinucleotide; FMNH Ϫ , reduced flavin mononucleotide; 4-NP, 4-nitrophenol; 4-CP, 4-chlorophenol; HQ, hydroquinone; BQ, benzoquinone; C 1 , the flavin reductase component of p-hydroxyphenylacetate 3-hydroxylase from A. buamannii; C 2 , the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase from A.

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Section: Catalytic Mechanisms Of Hadx Reductase and Hadb Reductase Thmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

Pimviriyakul

Chaiyen

2018

Journal of Biological Chemistry

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A Chemo‐Enzymatic Cascade for the Smart Detection of Nitro‐ and Halogenated Phenols

et al. 2019

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The flavin‐dependent monooxygenase, HadA, catalyzes the dehalogenation and denitration of the toxicants, nitro‐ and halogenated phenols, to benzoquinone. The HadA reaction can be applied in one‐pot reactions towards the de novo synthesis of d‐luciferin by coupling with d‐Cys condensation. d‐luciferin, a valuable chemical widely used in biomedical applications, can be used as a substrate for the reaction of firefly luciferase to generate bioluminescence. As nitro‐ and halogenated phenols are key indicators of human overexposure to pesticides and pesticide contamination, the technology provides a sensitive and convenient tool for improved biomedical and environmental detection at ppb sensitivity in biological samples without the requirement for any pre‐treatment. This dual‐pronged method combines the advantages of waste biodetoxification to produce a valuable chemical as well as a smart detection tool for environmental and biomedical detection.

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Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

et al. 2022

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D-Luciferin (D-LH 2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH 2 analogues with � 51 % yield. The method can synthesize the novel 5'-methyl-D-LH 2 and 4',5'-dimethyl-D-LH 2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH 2 emits brighter and longer wavelength light than the D-LH 2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

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

Abstract: Enzymes that are capable of detoxifying halogenated phenols (HPs) and nitrophenols (NPs) are valuable for bioremediation and waste biorefining.

Cited by 37 publications

References 83 publications

A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

A Chemo‐Enzymatic Cascade for the Smart Detection of Nitro‐ and Halogenated Phenols

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades

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