7e8p

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

spinqualitylabelsall models 7e8p, resolution 2.30Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 7e8p is a 4 chain structure with sequence from Ralstonia pickettii DTP0602. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.3Å Ligands: FDA, NPO Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function Q53008_RALPI Publication Abstract from PubMed HadA is a flavin-dependent monooxygenase catalyzing hydroxylation plus dehalogenation/denitration which is useful for biodetoxification and bio-detection. In this study, the X-ray structure of wild-type HadA (HadAWT) co-complexed with reduced FAD (FADH(-)) and 4-nitrophenol (4NP) (HadAWT-FADH(-)-4NP) was solved at 2.3 A 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, while the 4NP binding pocket contains the aromatic sidechain of Phe206, which provides pi-pi 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 pi-stacking (pi-pi-pi-pi) provided by two 4NP and two Phe441 from two subunits. This motif promotes 4NP binding in a non-productive dead-end complex which prevents C4a-hydroperoxy-FAD formation when HadA is pre-mixed with aromatic substrates. We also solved the structure of the HadAPhe441Val-FADH(-)-4NP complex at 2.3 A resolution. Although 4NP can still bind to this variant, the quadruple pi-stacking motif was disrupted. All HadAPhe441 variants lack substrate inhibition behavior, confirming that quadruple pi-stacking is a main cause of dead-end complex formation. Moreover, the activities of these HadAPhe441 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. Structural Insights into a Flavin-dependent Dehalogenase HadA Explain Catalysis and Substrate Inhibition via Quadruple pi-stacking.,Pimviriyakul P, Jaruwat A, Chitnumsub P, Chaiyen P J Biol Chem. 2021 Jul 9:100952. doi: 10.1016/j.jbc.2021.100952. PMID:34252455 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Monooxygenase 3D structures

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