7E8Q

Crystal structure of a Flavin-dependent Monooxygenase HadA F441V mutant complexed with reduced FAD and 4-nitrophenol

Summary for 7E8Q

• Entry DOI: 10.2210/pdb7e8q/pdb
• Related: 6JHM 7E8P
• Descriptor: Chlorophenol monooxygenase, DIHYDROFLAVINE-ADENINE DINUCLEOTIDE, P-NITROPHENOL, ... (4 entities in total)
• Functional Keywords: flavin monooxygenase, chlorophenol 4-monooxygenase, oxidoreductase
• Biological source: Ralstonia pickettii (Burkholderia pickettii)
• Total number of polymer chains: 4
• Total formula weight: 238285.39

Authors

Pimviriyakul, P.,Jaruwat, A.,Chitnumsub, P.,Chaiyen, P. (deposition date: 2021-03-02, release date: 2021-07-21, Last modification date: 2023-11-29)

Primary citation

Pimviriyakul, P.,Jaruwat, A.,Chitnumsub, P.,Chaiyen, P.
Structural insights into a flavin-dependent dehalogenase HadA explain catalysis and substrate inhibition via quadruple pi-stacking.
J.Biol.Chem., 297:100952-100952, 2021

PubMed Abstract: 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) co-complexed with reduced FAD (FADH) and 4-nitrophenol (4NP) (HadA-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-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 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 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.

PubMed: 34252455
DOI: 10.1016/j.jbc.2021.100952

Experimental method

X-RAY DIFFRACTION (2.3 Å)