Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase

Abstract

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s^-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Keywords: catalytic mechanism; diabetes; enzyme kinetics; flavin; flavoenzyme; flavoprotein; imidazole propionate; isotope effects; mutagenesis; oxidative half-reaction; reductase; stopped-flow; urocanate; urocanate reductase.

Figure 1

Summarized half-reactions of urocanate reductase (UrdA).

Figure 2

Comparison of active sites. Active site of (A) fumarate reductase in complex with fumarate (Protein Data Bank ID: 1D4E) and (B) urocanate reductase in complex with urocanate (Protein Data Bank ID: 6T87).

Figure 3

Dithionite reduction of UrdA’. UV–visible absorbance changes observed in the region of FAD absorbance after anaerobic reduction of 35 μM UrdA′-Fl_ox by sodium dithionite. The λ_max is 452 nm for oxidized enzyme. UrdA, urocanate reductase.

Figure 4

Reaction between UrdA′-Fl_redand urocanate.A, 15 μM UrdA′-Fl_ox was anaerobically reduced with dithionite, mixed with different concentrations of urocanate, and monitored for changes in FAD absorbance at pH 7, 4 °C using stopped-flow spectrophotometry. Each spectrum shows a different time point spanning from 1.6 ms to 0.2 s. The spectrum of UrdA′-Fl_red prior to mixing with urocanate is also shown for comparison. B, comparison of the absorbance spectra between UrdA′-Fl_ox (blue), UrdA′-Fl_red (black), and the intermediate formed within the dead time in the reaction between UrdA′-Fl_red and urocanate (red). C, absorbance trace overlay at 450 nm for the oxidation of wildtype UrdA′-Fl_red by different concentrations of urocanate at pH 7. The inset shows an absorbance trace overlay at 550 nm. D, k_obs values for the first and second phases of the reaction plotted against the urocanate concentration. UrdA, urocanate reductase.

Figure 5

Proposed kinetic model for the reaction between UrdA′-Fl_redand urocanate. UrdA, urocanate reductase.

Figure 6

Absorbance spectra of UrdA′-Fl_oxcomplexes at pH 7. Absorbance spectra for UrdA′-Fl_ox alone (blue), the UrdA′-Fl_ox–urocanate complex (red, λ_max = 454 nm), and the UrdA′-Fl_ox–imidazole propionate complex (green, λ_max = 455 nm). The UrdA′ concentration was 20 μM in all samples, and 200 μM ligand was added for the spectra of each complex. UrdA, urocanate reductase.

Figure 7

Isothermal titration calorimetry (ITC) measurement of urocanate binding to UrdA′ at 4$°\mathit{C}$. Upper panel, baseline-corrected thermogram for the titration. Lower panel, the black squares show the integrated heats for each injection, and the black line shows the fit to a one-site model. The fit provided a K_d of 160 ± 20 μM and a binding enthalpy (ΔH) of 6.4 ± 0.4 kcal/mol. UrdA, urocanate reductase.

Figure 8

pH dependence of the reaction between UrdA′-Fl_redand urocanate.A, stopped-flow absorbance traces for the reaction between wildtype UrdA′-Fl_red and saturating concentrations of urocanate at different pH values. Note the logarithmic time base. Reaction traces have been adjusted such that they all start at the same absorbance to facilitate comparison. Note that the UrdA′ concentration was not identical at each pH (varied from 9 to 16 μM used), and the kinetic amplitudes are therefore not directly comparable. B, pH dependence of the rate constant for flavin oxidation. Fitting the data to Equation 4 yielded apparent pKa values of 8.2 ± 0.3 and 9.4 ± 0.1 and k_ox values of 62 ± 3 s⁻¹ for the fully protonated state and 114 ± 13 s⁻¹ for the singly ionized state. C, k_obs values plotted against the concentration of urocanate for pH values 7, 10, 10.2, and, 10.5. Note the logarithmic y-axis. The k_obs values for pHs 10, 10.2, and 10.5 showed a hyperbolic dependence on urocanate concentration, which was fitted to Equation 3. The k_ox and K_d values from fitting the data can be found in Table 1. UrdA, urocanate reductase.

Figure 9

Stopped-flow absorbance traces for the oxidative half-reaction of UrdA′ mutants with saturating concentrations of urocanate at pH 7. Note the logarithmic time base. The k_ox and K_d values from fitting reaction traces can be found in Table 2. About 15 μM enzyme was used for each variant, and reaction traces have been adjusted such that they all start at the same absorbance to facilitate comparison. UrdA, urocanate reductase.

Figure 10

Plots of k_obsfor UrdA′ mutants with reduced affinity for urocanate.A, k_obs values plotted against the concentration of urocanate for the mutant Arg560Ala. The depicted graph exhibits a distinct hyperbolic relationship fitted to Equation 3, and this mutant necessitates considerably higher urocanate concentrations to achieve saturation of k_obs during the reaction. B, k_obs values plotted as a function of urocanate concentration for His520Ala and Phe245Ala also show a hyperbolic dependence, indicating higher K_d values for these mutants. UrdA, urocanate reductase.

Figure 11

Proposed mechanism of urocanate reduction by UrdA. The substrate binds in a twisted nonplanar conformation through interactions with charged residues. This arrangement facilitates hydride transfer from flavin N5 to urocanate C3, and Arg411 serves as the active site acid, donating a proton to urocanate C2. UrdA, urocanate reductase.