Mechanism of 4-Nitrophenol Oxidation in
            <i>Rhodococcus</i>
            sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression

Takeo, Masahiro; Murakami, Masumi; Niihara, Sanae; Yamamoto, Kenta; Nishimura, Munehiro; Kato, Dai‐ichiro; Negoro, Seiji

doi:10.1128/jb.00742-08

Cited by 75 publications

(53 citation statements)

References 47 publications

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“…5 and Table 2) also contributes to the general understanding of reactions of its homologs identified by SSN analysis (supplemental Fig. S1) (9,10,13,(33)(34)(35). These enzymes were identified in microbes that showed potential in bioremediation applications.…”

Section: Discussionmentioning

confidence: 99%

“…S1) showed that there are 2,839 enzymes in which the sequences have Ͼ34% identity with HadA, and only 11 of these 2,839 enzymes were reviewed by UniProt. The enzymes identified to be homologous to HadA were mostly flavin-dependent monooxygenases that incorporate a hydroxyl group into phenols such as CPs or nitrophenols (9,10,(33)(34)(35). As these enzymes showed potential applications in bioremediation of toxic compounds, the understanding of kinetic mechanisms associated with a flavin-dependent dechlorinase (HadA) reported herein should also be useful for giving an overall picture about the reaction mechanism of these enzymes in general.…”

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confidence: 99%

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Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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Edited by F. Peter GuengerichThe accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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“…(182), Rhodococcus opacus SAO101 (100), and Rhodococcus sp. PN1 (192) all use a monooxygenase that directly oxidizes 4-nitrophenol to benzoquinone, followed by reduction to hydroquinone (182). The pathways converge at ring cleavage, where 1,2,4-trihydroxybenzene and hydroquinone are each converted into maleylacetate, which is then reduced to ␤-ketoadipate and converted to TCA cycle intermediates.…”

Section: Pathways For Nitrophenol Catabolismmentioning

confidence: 99%

Nitroaromatic Compounds, from Synthesis to Biodegradation

Parales

2010

Microbiol Mol Biol Rev

744

470

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SUMMARY Nitroaromatic compounds are relatively rare in nature and have been introduced into the environment mainly by human activities. This important class of industrial chemicals is widely used in the synthesis of many diverse products, including dyes, polymers, pesticides, and explosives. Unfortunately, their extensive use has led to environmental contamination of soil and groundwater. The nitro group, which provides chemical and functional diversity in these molecules, also contributes to the recalcitrance of these compounds to biodegradation. The electron-withdrawing nature of the nitro group, in concert with the stability of the benzene ring, makes nitroaromatic compounds resistant to oxidative degradation. Recalcitrance is further compounded by their acute toxicity, mutagenicity, and easy reduction into carcinogenic aromatic amines. Nitroaromatic compounds are hazardous to human health and are registered on the U.S. Environmental Protection Agency's list of priority pollutants for environmental remediation. Although the majority of these compounds are synthetic in nature, microorganisms in contaminated environments have rapidly adapted to their presence by evolving new biodegradation pathways that take advantage of them as sources of carbon, nitrogen, and energy. This review provides an overview of the synthesis of both man-made and biogenic nitroaromatic compounds, the bacteria that have been identified to grow on and completely mineralize nitroaromatic compounds, and the pathways that are present in these strains. The possible evolutionary origins of the newly evolved pathways are also discussed.

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“…More recently, a twocomponent PNP hydroxylase from Rhodococcus sp. strain PN1 that catalyzes the hydroxylation of PNP to form 4-nitrocatechol was characterized (32). Although the PNP catabolic genes for the hydroquinone pathway were reported to have been cloned from Pseudomonas sp.…”

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confidence: 99%

Identification and Characterization of Catabolicpara-Nitrophenol 4-Monooxygenase andpara-Benzoquinone Reductase fromPseudomonassp. Strain WBC-3

et al. 2009

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Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N.

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Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression

Abstract: 4-Nitrophenol (4-NP

Cited by 75 publications

References 47 publications

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Nitroaromatic Compounds, from Synthesis to Biodegradation

Identification and Characterization of Catabolicpara-Nitrophenol 4-Monooxygenase andpara-Benzoquinone Reductase fromPseudomonassp. Strain WBC-3

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