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

Panu Pimviriyakul,Aritsara Jaruwat,Penchit Chitnumsub,Pimchai Chaiyen

doi:10.1016/j.jbc.2021.100952

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 (HadAWT) co-complexed with reduced FAD (FADH–) and 4-nitrophenol (4NP) (HadAWT−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 HadAPhe441Val−FADH–−4NP complex at 2.3-Å resolution. Although 4NP can still bind to this variant, the quadruple π-stacking motif was disrupted. All HadAPhe441 variants lack substrate inhibition behavior, confirming that quadruple π-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.

Highlights

Halogenated phenols (HPs) and nitrophenol (NP) are persistent pollutants resulting from anthropogenic activities including the use of agro- and household chemicals such as pesticides, herbicides, and flame retardants
The co-complex with FADH– and 4NP allowed identification of four binding sites for ligands in which strong electron densities could be observed for all four FADH– molecules, whereas only one 4NP molecule could be clearly seen in the expected pocket of subunit A (Fig. 2B, Inset)
The key difference between the HadAWT−FADH–−4NP structure reported here and the previously reported apo-HadAWT structure is the loop of residues 157 to 170, which assumes a distinct structure in the ternary complex, whereas it is disordered in the apo-HadAWT

Summary

Introduction

Halogenated phenols (HPs) and nitrophenol (NP) are persistent pollutants resulting from anthropogenic activities including the use of agro- and household chemicals such as pesticides, herbicides, and flame retardants. The hydrophobic environment found in the HadA active site is similar to the environment of an empty pocket speculated to be the phenol-binding pocket in apoenzyme structures of dehalogenating flavindependent enzymes previously crystallized (Fig. S6) [15,16,17].

Results

Conclusion