A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin-dependent enzymes

Abstract

Halogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the had operon from the bacterium Ralstonia pickettii DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH^-)-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH^- for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH^- for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH^- and that HadX can transfer FADH^- from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH^- for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, i.e. HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications.

Keywords: bioremediation; biotechnology; enzyme kinetics; enzyme mechanism; flavin; flavin adenine dinucleotide (FAD); flavoenzyme; halogenated phenol; nitrophenol; reductase.

Figure 1.

Proposed pathway for phenol degradation by the had operon. Enzyme abbreviations: HadA, dechlorinating and denitrating flavin-dependent monooxygenase; HadC, hydroxyquinol 1,2-dioxygenase; HadD, maleylacetate reductase. *, the second hydroxylation and group elimination by HadA can occur if Y is halide or nitro groups.

Figure 2.

HadB can prevent quinone polymerization. A–F, absorption spectra of multiple turnover reactions of HadA in the absence (A–C) and presence of HadB (1 μm) (D–F) using 2,4-DCP, 2,4,5-TCP, and 2,4,6-TCP as substrates. The FADH⁻ generating system employed consisted of C₁ reductase (1 μm), FAD (10 μm), Glc-6-P dehydrogenase (0.5 unit/ml), and Glc-6-P (1 mm).

Figure 3.

Transient kinetics of the reductive and oxidative half-reaction of HadB. A and B, kinetic traces of the reactions of anaerobic HadB_FMN (12 μm) mixing with (A) NADH or (B) NADPH (0.05–3.2 mm) in 50 mm NaP_i, pH 7.0, at 4 °C. Inset in B shows a plot between the observed rate constant (k_obs) of the first phase of the reaction versus NADPH concentrations which yields a hyperbolic relationship. C, kinetic traces of the reactions of anaerobic HadB_FMNH− (12.5 μm) mixing with various concentrations of menadione (50–200 μm) in 50 mm NaP_i, pH 7.0, at 4 °C (solid lines) and reactions of HadB_FMNH− (12.5 μm) mixed with O₂ (0.13 mm) in 50 mm NaP_i, pH 7.0, at 25 °C (dashed lines) were obtained by monitoring the absorbance of the reactions at 456 nm.

Figure 4.

Proposed reaction mechanism of HadB quinone reductase. k_red and k_*^red represent the rate constants of HadB_FMN and HadB_FMN^* reduction by NAD(P)H, respectively. k_ox represents the rate constant of electron transfer from HadB_FMNH− to an electron acceptor such as menadione.

Figure 5.

Transient kinetics of the reductive half-reaction of HadX. Kinetic traces of reactions of anaerobic HadX_FAD (15 μm) mixed with various concentrations of NADH (0.05–3.2 mm) in 50 mm NaP_i, pH 7.0, at 4 °C were obtained by monitoring the absorbance at 456 (solid lines) and 600 nm (dashed lines) to detect flavin reduction of HadB and formation of the charge-transfer complex, respectively. The inset is a plot between the observed rate constants of the first phase versus NADH concentration.

Figure 6.

Proposed reaction mechanism of HadX flavin reductase. k_red,1 and k_red,2 represent the rate constants of HadX_FAD and HadX_FAD^* reduction by NADH, respectively. K_D,NADH1 and K_D,NADH2 represent dissociation constants for bindings of NADH to HadX_FAD and HadX_FAD^*, respectively. k_ox,1, k_ox,2, and k_ox,3 represent the rate constants of free FADH⁻, HadX_FADH−, and HadX_FADH−^* reacting with oxygen, respectively.

Figure 7.

Transient kinetics of the reaction of HadX_FADH− and oxygen. Kinetic traces of anaerobic HadX_FADH− (15 μm) mixed with various concentrations of oxygen (0.13, 0.31, and 0.61 mm) in 50 mm NaP_i, pH 7.0, at 25 °C were obtained by monitoring the absorbance at 456 nm. Inset shows a plot of the observed rate constants of the 1st phase (circle line), 2nd phase (square line), and 3rd phase (diamond line) versus oxygen concentration, yielding linear relationships with bimolecular rate constants of 2.5 ± 0.2 × 10⁴, 4.3 ± 0.1 × 10³, and 1.2 ± 0.1 × 10³ m⁻¹ s⁻¹, respectively.

Figure 8.

Kinetics of the transfer of FADH⁻ from HadX to HadA. Anaerobic solutions of HadX_FADH− (15 μm) were mixed against aerobic HadA (0.1 mm) in 50 mm NaP_i, pH 7.0, at 25 °C. Kinetic traces (solid line) detected at wavelengths of (A) 380 and (B) 456 nm are compared with kinetic traces of aerobic HadA mixed with free FADH⁻ (dashed line) previously reported from Ref. . Inset in B are the spectra of three flavin species including the HadX_FADH− (dashed line), the intermediate obtained at 10 s after the reaction started (solid line with filled circles), and a mixture of HadA and HadX_FADH− (solid line).

Figure 9.

Transfer of FADH⁻ from HadX reductase to HadA monooxygenase.

Figure 10.

Determination of a rate constant of FADH⁻ transfer from HadX to C₂ monooxygenase. Double-mixing stopped-flow experiments were carried out by mixing a solution of HadX_FADH− (15 μm) with C₂ (25 μm) under anaerobic conditions in the first mixing. The mixing was prolonged at different age times (0.01–4 s) prior to mixing with a buffer containing oxygen (0.61 mm) under the second mixing. Absorption changes at wavelength 380 nm were measured to monitor the formation of C4a-hydroperoxy-FAD. Inset is a plot of ΔA₃₈₀ at t = 0.02 s (the amount of C4a-hydroperoxy-FAD intermediate formed) versus age times to obtain the rate constant of the FADH⁻ transfer from HadX_FADH− to C₂ monooxygenase as 7.0 ± 0.6 s⁻¹.

Figure 11.

Biodegradation of 4-NP and 4-CP by cascade reactions of HadA monooxygenase, HadX and HadB reductases. Multiple turnover reactions of HadA consisted of (A) 4-NP (100 μm) or (B) 4-CP (100 μm), Glc-6-P (1 mm), Glc-6-P dehydrogenase (0.5 unit/ml), NAD⁺ (5 μm), HadA (10 μm), with different combinations of reductases in 50 mm NaP_i, pH 8.0, at room temperature. Reactions were quenched at various times by adding an equal volume of acetonitrile. Samples were diluted 3-fold in 50 mm NaP_i, pH 8.0, and ascorbic acid (0.5 mm) to convert all BQ products to HQ for more convenient analysis by HPLC/DAD. Blue circle lines are the reactions containing HadX (1 μm). Red square lines are the reactions containing HadX (1 μm) and HadB (1 μm). Green diamond lines are the reactions containing HadX (1 μm) and ascorbic acid (1 mm). Purple triangle lines are the reactions without any reductase.

Figure 12.

Overall mechanisms and steps involved in the detoxification of halogenated phenols and nitrophenols by three flavin-dependent enzymes in the had operon.