Identification of the upstream 4-chlorophenol biodegradation pathway using a recombinant monooxygenase from Arthrobacter chlorophenolicus A6

Cho, Su Yeon; Kwean, Oh Sung; Yang, Jun Won; Cho, Wooyoun; Kwak, Seon-Yeong; Park, Sungyoon; Lim, Yejee; Kim, Han Soo

doi:10.1016/j.biortech.2017.05.006

Cited by 26 publications

(10 citation statements)

References 35 publications

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“…To analyze whether the nature of the amino acids found in the FADH – -binding pocket of the ternary complex structure of HadA is also common in other group D monooxygenases, amino acid sequences and structures of these enzymes were analyzed. Sequences of (1) flavin-dependent monooxygenases catalyzing hydroxylation plus dehalogenation and denitration activities (HadA monooxygenase from R. pickettii [this study]); (2) flavin-dependent monooxygenases catalyzing hydroxylation plus dehalogenation ( Re TcpA from R. eutropha JMP134 ( 15 , 28 ), Cn TcpA from Cupriavidus nantongensis X1 T ( 29 ), TftD from B. cepacia AC1100 ( 17 , 30 ), CphC-I from Arthrobacter chlorophenolicus A6 ( 31 ), DcmB1 from Rhodococcus sp. JT-3 ( 32 ), and HnpA from Cupriavidus sp.…”

Section: Resultsmentioning

confidence: 99%

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

Pimviriyakul

Jaruwat

Chitnumsub

et al. 2021

Journal of Biological Chemistry

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HadA is a flavin-dependent monooxygenase catalyzing hydroxylation plus dehalogenation/denitration, which is useful for biodetoxification and biodetection. In this study, the X-ray structure of wild-type HadA (HadA WT ) co-complexed with reduced FAD (FADH – ) and 4-nitrophenol (4NP) (HadA WT −FADH – −4NP) was solved at 2.3-Å resolution, providing the first full package (with flavin and substrate bound) structure of a monooxygenase of this type. Residues Arg101, Gln158, Arg161, Thr193, Asp254, Arg233, and Arg439 constitute a flavin-binding pocket, whereas the 4NP-binding pocket contains the aromatic side chain of Phe206, which provides π-π stacking and also is a part of the hydrophobic pocket formed by Phe155, Phe286, Thr449, and Leu457. Based on site-directed mutagenesis and stopped-flow experiments, Thr193, Asp254, and His290 are important for C4a-hydroperoxyflavin formation with His290, also serving as a catalytic base for hydroxylation. We also identified a novel structural motif of quadruple π-stacking (π-π-π-π) provided by two 4NP and two Phe441 from two subunits. This motif promotes 4NP binding in a nonproductive dead-end complex, which prevents C4a-hydroperoxy-FAD formation when HadA is premixed with aromatic substrates. We also solved the structure of the HadA Phe441Val −FADH – −4NP complex at 2.3-Å resolution. Although 4NP can still bind to this variant, the quadruple π-stacking motif was disrupted. All HadA Phe441 variants lack substrate inhibition behavior, confirming that quadruple π-stacking is a main cause of dead-end complex formation. Moreover, the activities of these HadA Phe441 variants were improved by ⁓20%, suggesting that insights gained from the flavin-dependent monooxygenases illustrated here should be useful for future improvement of HadA’s biocatalytic applications.

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Section: Resultsmentioning

confidence: 99%

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

Pimviriyakul

Jaruwat

Chitnumsub

et al. 2021

Journal of Biological Chemistry

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“…Oxidative dehalogenases are usually encoded by operons found in aerobic bacteria such as the tft cluster from Burkholderia cepacia AC1100, 26 , 27 the tcp cluster from Ralstonia eutropha JMP134, 28 , 29 the had cluster from Ralstonia pickettii DTP0602, 30 – 33 the pcp cluster from Sphingobium chlorophenolicum , 34 – 37 and the cph cluster from Arthrobacter chlorophenolicus A6. 38 These enzymes can dechlorinate CPs and incorporate hydroxyl groups. They are mostly single-component or two-component flavin-dependent monooxygenases.…”

Section: Introductionmentioning

confidence: 99%

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

2018

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“…; Cho et al . ). The Kocuria genus includes several halotolerant and phenol degrading strains (Takarada et al .…”

Section: Discussionmentioning

confidence: 97%

“…These aerobic micro-organisms are metabolically diverse and possess the ability to survive under stressful conditions for extended periods of time (Mongodin et al 2006). Members of the Arthrobacter genus have presented the ability to degrade aromatic compounds like 4-nitrophenol and 4-chlorophenol (Qu et al 2012;Cho et al 2017). The Kocuria genus includes several halotolerant and phenol degrading strains (Takarada et al 2008).…”

Section: Discussionmentioning

confidence: 99%

Analysis of a phenol-adapted microbial community: degradation capacity, taxonomy and metabolic description

et al. 2019

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Aims In this study, the ability of the consortium MR‐01 to degrade phenol was determined. The effects of this chemical on the taxonomy and the metabolic behaviour were analysed through metagenomics. Methods and Results Consortium MR‐01 was acclimated in a sublethal concentration of phenol. After this process, the capacity to degrade this molecule was analysed. Results showed that degradation increased with the increment of the initial phenol concentration. Metagenomic analysis indicates that the consortium metabolized phenol under aerobic conditions using phenol 2‐monooxygenase and the meta‐cleavage pathway. Sequence of the enzymes involved in the phenol degradation was ascribed to the Actinomycetales and Chloroflexales orders, with relative abundances <1%. The most abundant genera were part of the Sphingomonadales order; however, the role of these species in the consortium is not clear. Conclusions Consortium MR‐01 degrades efficiently high concentrations of phenol. The participation of extremophiles in the degradation process and the emergence of beneficial metabolic dependencies between the community members are some of the strategies used by the consortium to survive and develop under harsh environmental conditions. Significance and Impact of the Study This is one of the few studies describing the taxonomy and metabolic profile of a phenol degrading consortium.

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Identification of the upstream 4-chlorophenol biodegradation pathway using a recombinant monooxygenase from Arthrobacter chlorophenolicus A6

Cited by 26 publications

References 35 publications

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking

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

Analysis of a phenol-adapted microbial community: degradation capacity, taxonomy and metabolic description

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