Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1

Takeo, Masahiro; Yasukawa, Takeshi; Abe, Yoshikatsu; Niihara, Sanae; Maeda, Yoshimichi; Negoro, Seiji

doi:10.1016/s1389-1723(03)80119-6

Cited by 47 publications

(33 citation statements)

References 21 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: 88%

“…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: 97%

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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: 88%

mentioning

confidence: 97%

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Pimviriyakul

Thotsaporn

Sucharitakul

et al. 2017

Journal of Biological Chemistry

108

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“…In the phylogenetic tree, both the NpcB and NpcA proteins are located in branches that differ from those of the 4-NP monooxygenase protein NphA2 (reductase) and NphA1 (oxygenase) of Rhodococcus sp. strain PN1 (26). This might be reflective of enzyme function.…”

Section: Discussionmentioning

confidence: 98%

“…strain PN1. The gene products NphA1 and NphA2 are reported to convert 4-NP to 4-NCA, but the genes involved in the further degradation of 4-NCA and its metabolites from strain PN1 have not been reported to date (26). Therefore, more information about the catabolic genes involved in 4-NP metabolism is still needed, and in particular, information about those genes that act on the hydroxyquinol pathway is considered to be highly desirable.…”

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confidence: 99%

A Novel p -Nitrophenol Degradation Gene Cluster from a Gram-Positive Bacterium, Rhodococcus opacus SAO101

2004

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p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.

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“…Several bacterial strains belonging to diverse taxonomic groups have been isolated and characterized for degradation of the above compounds [5,6]. The molecular regulation of some of these degradation pathways have also been elucidated in detail [7][8][9]. Aromatic ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes that catalyze aromatic ring cleavage for complete degradation of these compounds.…”

Section: Introductionmentioning

confidence: 99%

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

Paul¹,

Rastogi²,

Krauß³

et al. 2008

Indian J Microbiol

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Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono-phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great signifi cance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.

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Cloning and characterization of a 4-nitrophenol hydroxylase gene cluster from Rhodococcus sp. PN1

Cited by 47 publications

References 21 publications

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA

A Novel p -Nitrophenol Degradation Gene Cluster from a Gram-Positive Bacterium, Rhodococcus opacus SAO101

Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria

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