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

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(–)...

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Autores principales: Pimviriyakul, Panu, Jaruwat, Aritsara, Chitnumsub, Penchit, Chaiyen, Pimchai
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342789/
https://www.ncbi.nlm.nih.gov/pubmed/34252455
http://dx.doi.org/10.1016/j.jbc.2021.100952
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author Pimviriyakul, Panu
Jaruwat, Aritsara
Chitnumsub, Penchit
Chaiyen, Pimchai
author_facet Pimviriyakul, Panu
Jaruwat, Aritsara
Chitnumsub, Penchit
Chaiyen, Pimchai
author_sort Pimviriyakul, Panu
collection PubMed
description 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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spelling pubmed-83427892021-08-11 Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking Pimviriyakul, Panu Jaruwat, Aritsara Chitnumsub, Penchit Chaiyen, Pimchai J Biol Chem Research Article 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. American Society for Biochemistry and Molecular Biology 2021-07-10 /pmc/articles/PMC8342789/ /pubmed/34252455 http://dx.doi.org/10.1016/j.jbc.2021.100952 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Pimviriyakul, Panu
Jaruwat, Aritsara
Chitnumsub, Penchit
Chaiyen, Pimchai
Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title_full Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title_fullStr Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title_full_unstemmed Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title_short Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple π-stacking
title_sort structural insights into a flavin-dependent dehalogenase hada explain catalysis and substrate inhibition via quadruple π-stacking
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8342789/
https://www.ncbi.nlm.nih.gov/pubmed/34252455
http://dx.doi.org/10.1016/j.jbc.2021.100952
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