Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase

Abstract

Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N(5)-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O(2). The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP(+) (t(½) = 33 min, 25 °C, pH 8). NADP(+) therefore is a likely regulator of O(2) and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N(5)-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.

SCHEME 1.

Fusarinine biosynthesis, with initiating step catalyzed by OMO.

SCHEME 2.

Kinetic mechanism for OMO.

COMPLEX 1

FIGURE 1.

Reduction of OMO by NADPH. The reaction mixture contained 10 μm enzyme at 25 °C. Absorbance was measured in photomultiplier mode at 450 nm, and traces were fit to the sum of two exponentials. Observed rates were 0.58, 0.67, and 0.60 s⁻¹ at 10 (long dashed line), 100 (solid line), and 200 (short dashed line) μm NADPH, where the trace at 100 μm NADPH contained 5 mm l-ornithine.

FIGURE 2.

A, formation of the C4a-hydroperoxyflavin·NADP⁺ complex. Spectra are shown at 0.004, 0.071, 0.139, 0.307, 0.577, 0.960, and 2.355 s after mixing the NADPH-reduced enzyme with air-saturated buffer (final [O₂] = 0.130 mm) in the stopped-flow instrument. Inset, oxygen dependence of the rate of C4a-hydroperoxyflavin formation in the absence of l-Orn. C4a-hydroperoxide formation was monitored at 370 nm under pseudo-first order conditions with varying [O₂]. Values for k_obs determined from fitting single exponentials to the data are plotted versus [O₂]. The second order rate constant determined from this plot was 2.5 × 10⁴ m⁻¹ s⁻¹. Reaction mixtures contained 10 μm reduced enzyme·NADP⁺ at 25 °C. B, conversion of the C4a-hydroperoxyflavin·NADP⁺ complex to oxidized FAD. Spectra at 2.63, 11.25, 18.6, 29.4, 60.3, and 142.5 s after mixing from the experiment in Fig. 2 are shown.

FIGURE 3.

A, formation of the C4a-hydroperoxyflavin·NADP⁺ complex in the presence of l-Orn. Spectra are shown at 0.004, 0.019, 0.034, 0.049, 0.071, 0.079, 0.011, and 0.026 s after mixing the NADPH-reduced enzyme with air-saturated buffer containing 5 mm l-Orn (final [O₂] = 0.130 mm). The reaction mixture contained ∼10 μm reduced enzyme·NADP⁺ complex at 25 °C. Inset, oxygen dependence of the rate of C4a-hydroperoxyflavin formation in the presence of saturating l-Orn. The conversion of the reduced enzyme·NADP⁺ complex to the C4a-hydroperoxyflavin was monitored at 370 nm in the presence of 5 mm l-Orn and varying (pseudo-first order) [O₂]. Values for k_obs were determined from fits of single exponential curves to the data and plotted versus [O₂]. The second order rate constant determined from this plot was 1.3 × 10⁵ m⁻¹s⁻¹. B, kinetic traces illustrating the O₂ dependence of the reaction of reduced enzyme·NADP⁺ complex with O₂ in the presence of l-Orn. Absorbance traces at 370 nm show the reduced enzyme·NADP⁺ complex reacting with varying [O₂] to form the C4a-hydroperoxyflavin. Final oxygen concentrations were 0.06, 0.13, 0.3, and 0.6 mm. The kinetic trace at 450 nm illustrates the formation of the oxidized FAD that occurs as the C4a-hydroperoxyflavin disappears, a reaction that shows no dependence on [O₂]. C, dependence of the rate of C4a-hydroperoxide formation on [l-Orn] at fixed oxygen. The conversion of reduced enzyme·NADP⁺ complex to the C4a-hydroperoxyflavin was monitored at 370 nm at various (pseudo-first order) [l-Orn] at fixed/saturating O₂. Rate constants were determined from single exponential fits to the first portion of the curves. The apparent K_d and maximal k_obs determined from this plot were 680 μm and 82 s⁻¹. Each reaction mixture contained 10 μm reduced enzyme·NADP⁺ complex with 0.3 mm O₂ at 25 °C.

FIGURE 4.

A, conversion of the C4a-hydroperoxyflavin·NADP⁺ complex to oxidized OMO in the presence of l-Orn. 10 μm reduced enzyme·NADP⁺ complex containing 5 mm l-Orn was mixed with air-saturated buffer (final [O₂] = 0.3 mm at 25 °C). The C4a-hydroperoxyflavin species (λ_max = 370 nm) formed and subsequently converted to the oxidized flavin. Only the spectra showing the latter conversion are shown (recorded 0.04, 0.30, 0.50, 1.10, 2.00, and 7.00 s after mixing). B, dependence of the conversion of the C4a-hydroperoxide to oxidized FAD on l-Orn at fixed/saturating O₂. The conversion of the C4a-hydroperoxyflavin to the oxidized FAD shown in A was monitored at 450 nm at various (pseudo-first order) l-Orn concentrations. Values for k_obs were determined from single exponential fits to the curves. The apparent K_d and maximal k_obs determined from this plot were 2.2 mm and 1.8 s⁻¹.

FIGURE 5.

A, conversion of a preformed C4a-hydroperoxyflavin·NADP⁺ complex to oxidized OMO in the presence of l-Orn. 10 μm reduced enzyme·NADP⁺ complex in the absence of l-Orn was mixed with air-saturated buffer (final [O₂] = 0.3 mm at 25 °C) and aged until the C4a-hydroperoxyflavin intermediate fully formed (5 s). The preformed intermediate was then mixed with 5 mm l-Orn in the same buffer. Spectra shown were recorded 0.161, 0.352, 0.487, 0.622, 1.050, and 7.125 s after the second mixing. B, kinetic traces showing the reaction of C4a-hydroperoxide to oxidized FAD at 450 nm. Reactions were monitored at 450 nm. Final [l-Orn] = 0.0625, 0.25, 0.5 1.25, 2.5, 5.0, and 25.0 mm. C, dependence of the conversion of the C4a-hydroperoxide to oxidized FAD on l-Orn at fixed/saturating O₂. The conversion of the C4a-hydroperoxyflavin to the oxidized FAD shown in A was monitored at 450 nm at various (pseudo-first order) l-Orn concentrations. Values for k_obs were determined from single exponential fits to the curves. The apparent K_d and maximal k_obs determined from this plot were 2.3 mm and 2.5 s⁻¹, respectively.

FIGURE 6.

A, kinetic traces illustrating the reaction of a preformed C4a-hydroperoxide with variable concentrations of l-Orn. The C4a-hydroperoxide was generated by mixing 10 μm reduced enzyme·NADP⁺ complex (in the absence of l-Orn) with air-saturated buffer (final [O₂] = 0.3 mm, 25 °C) and aged 5 s. It was then mixed with various l-Orn concentrations in the same buffer. The data measured at 393 nm are shown, illustrating three consecutive exponential processes. Final [l-Orn] for the traces shown is 1.25, 2.5, 5.0, and 25.0 mm. B, singular value decomposition of the complete set of data shown in Fig. 5A, using the rate constants fit to the three kinetic phases highlighted in A. Three constituent spectra were determined, resembling previously measured spectra for C4a-hydroperoxide (dotted line), C4a-hydroxide (solid line), and oxidized FAD (dashed line).