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

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_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.

Keywords: biodegradation; crystal structure; enzyme kinetics; flavin-dependent monooxygenase/dehalogenase; halogenated phenol; inhibition mechanism; nitrophenol; quadruple π-stacking; site-directed mutagenesis.

Figure 1

The overall reaction mechanism of HadA monooxygenase (6, 7, 8).

Figure 2

Crystal structure of the HadA_WT–FADH^––4NP complex.A, tetrameric quaternary structure of HadA monooxygenase co-complexed with FADH^– and 4NP. B, three domains of the HadA subunit include the N-terminal domain (green part), the β-sheet domain (orange part), and C-terminal domain (purple part). The flavin-binding loop (residues 157–170) is labeled in red. Inset in (B) is an electron density map of FADH^– and 4NP bound in the active site. 4NP, 4-nitrophenol.

Figure 3

Binding interactions of FADH^–and 4NP within the active site of HadA_WT.A, amino acid residues and interactions at the binding pockets of 4NP, and adenosine and isoalloxazine moieties of FADH^–. FADH^– is shown in yellow, whereas 4NP is shown in green. Pink residues indicate amino acids from subunit A, whereas cyan residues indicate amino acids from subunit B. B, a simple scheme to illustrate interactions of 4NP and FADH^– with residues in the active site of HadA_WT. 4NP, 4-nitrophenol.

Figure 4

Relative 4NP consumption activities of HadA variants. Multiple turnover reactions consisting of 4-nitrophenol or 4NP (50 μM), Glc-6-P (1 mM), Glc-6-PD (0.5 unit/ml), NAD⁺ (5 μM), HadX (1 μM), HadA variants (10 μM) in 20 mM Hepes pH 7.5 were carried out. UV-visible spectra were monitored to observe the decrease of absorption around the 400-nm region due to loss of the 4NP substrate. 4NP consumption activity of HadA_WT was set to 100% for comparison with Thr193, Asp254, His290, Phe206, and Phe286 variants.

Figure 5

Transient kinetics of HadA variants. Rapid kinetics of reactions of HadA_variant–FADH^– (75 and 25 μM, respectively) mixing with an aerobic solution of 4-chlorophenol (0.5 mM 4-chlorophenol and 0.13 mM O₂) using the single mixing mode of a stopped-flow apparatus. Absorption changes at wavelengths 380 nm (solid line) and 450 nm (dashed line) were monitored to observe the C4a-hydroperoxy-FAD intermediate and oxidized FAD, respectively. C4a-hydroperoxy-FAD absorbs mainly at A₃₈₀, whereas oxidized FAD absorbs at both wavelengths. HadA variants are (A) HadA_WT, (B) HadA_Thr193Ala, (C) HadA_Thr193Val, (D) HadA_Thr193Ser, (E) HadA_Asp254Ala, (F) HadA_Asp254Asn, (G) HadA_His290Ala, (H) HadA_His290Cys, and (I) HadA_His290Asn. Inset in (A) and (I) are spectra of the C4a-hydroperoxy-FAD intermediate detected at 10 s of reaction time.

Figure 6

Conserved amino acid residues of group D two-component monooxygenases. Important residues in HadA_WT including the FADH^–-binding region, substrate-binding region, and subunit interface were analyzed by Clustal omega (EMBL-EBI). The figure was generated by WebLogo.

Figure 7

Proposed reaction mechanisms of HadA monooxygenase with halogenated phenols (HPs) or nitrophenol (NP) bound in its active site.

Figure 8

Intermolecular binding of 4-nitrophenol at the interface of HadA dimer.A, a quadruple π-stacking, an unusual π-π-π-π interaction formed by two 4-ntirophenol (4NP) substrates sandwiched with two Phe441 residues of the HadA_WT dimer observed in the HadA_WT–FADH^––4NP structure. B, disruption of a quadruple π-stacking in the HadA_Phe441Val–FADH^––4NPstructure. The lower panel shows side chains of residues from subunit C (yellow) and subunit D (blue) residing within 4-Å distances from the two 4NP molecules bound in the cavity of the subunit interface.

Figure 9

Regulation of dead-end complex formation by Phe441. An anaerobic solution of FADH^– (25 μM) was rapidly mixed with an air saturated (A) HadA_WT, (B) HadA_Phe441Val, or (C) HadA_Phe441Leu (75 μM) that was preincubated with 4CP (0.1–6.4 mM) in 20 mM Hepes pH 7.5. Absorption changes at wavelengths 380 (blue solid line) and 450 nm (red dash line) were monitored to detect formation of the C4a-hydroperoxy-FAD intermediate and oxidized FAD, respectively. C4a-hydroperoxy-FAD absorbs mainly at 380 nm, whereas oxidized FAD absorbs at both wavelengths. D, HQ product formation at various concentrations of 4-chlorophenol (4CP) catalyzed by HadA_WT (black circle), HadA_Phe441Val (blue square), and HadA_Phe441Leu (red diamond).

Figure 10

Efficiency of 4-nitrophenol utilization by HadA_Phe441variants. Multiple turnover reactions consisting of 4NP (100 μM), Glc-6-P (1 mM), Glc-6-PD (0.5 unit/ml), NAD⁺ (5 μM), HadX (1 μM), and HadA_variants (0.1 μM) in 20 mM Hepes pH 7.5 were carried out. The molar ratio of 4NP:HadA used is 1000:1. Plots comparing the percentage of 4NP remaining in the denitrification reactions of HadA_WT (black line), HadA_Phe441Val (blue line), HadA_Phe441Leu (green line), and HadA_Phe441Ile (red line) versus time. Inset is a plot of rates of 4NP consumption by various types of HadA. 4NP, 4-nitrophenol.