Flavin oxidation in flavin-dependent N-monooxygenases

Abstract

Siderophore A (SidA) from Aspergillus fumigatus is a flavin-containing monooxygenase that hydroxylates ornithine (Orn) at the amino group of the side chain. Lysine (Lys) also binds to the active site of SidA; however, hydroxylation is not efficient and H₂ O₂ is the main product. The effect of pH on steady-state kinetic parameters was measured and the results were consistent with Orn binding with the side chain amino group in the neutral form. From the pH dependence on flavin oxidation in the absence of Orn, a pK_a value >9 was determined and assigned to the FAD-N5 atom. In the presence of Orn, the pH dependence displayed a pK_a value of 6.7 ±0.1 and of 7.70 ±0.10 in the presence of Lys. Q102 interacts with NADPH and, upon mutation to alanine, leads to destabilization of the C4a-hydroperoxyflavin (FAD_OOH ). Flavin oxidation with Q102A showed a pK_a value of ~8.0. The data are consistent with the pK_a of the FAD N5-atom being modulated to a value >9 in the absence of Orn, which aids in the stabilization of FAD_OOH . Changes in the FAD-N5 environment lead to a decrease in the pK_a value, which facilitates elimination of H₂ O₂ or H₂ O. These findings are supported by solvent kinetic isotope effect experiments, which show that proton transfer from the FAD N5-atom is rate limiting in the absence of a substrate, however, is significantly less rate limiting in the presence of Orn and or Lys.

Keywords: flavin-dependent monooxygneases; hydroperoxyflavin; ornithine hydroxylase; oxidation; pH profile; siderophore; solvent kinetic isotope effect.

Figure 1

(A) Kinetic mechanism for the reaction catalyzed by SidA. The enzyme begins with the flavin in the oxidized form (FAD_OX). NADPH binds and reduces the flavin (FAD_red). The reduced FAD/NADP⁺ complex reacts with oxygen, forming the C4a‐hydroperoxyflavin (FAD_OOH). This is followed by Orn binding and hydroxylation at the Orn N5‐atom. The next step is release of H₂O from the hydroxyflavin (FAD_OH). Release of products is shown to occur in one step. (B) Scheme of the FAD_OOH, NADP⁺, Orn complex obtained from previous molecular dynamics simulation and density functional theory studies.7, 8

Figure 2

Effect of pH on the steady‐state oxygen consumption activity of SidA. (A) Effect of pH on the k _cat value. (B) Effect of pH on the k _cat/K _m value. The pH profiles were analyzed using Eq. (3) and pK _a values of 7.0 ±0.2 and 8.0 ±0.2 were obtained for k _cat and k _cat/K _m, respectively. (C) Changes in the K _m value as a function of pH. Values used in all plots are listed in Table S1. The line on panel C connects the points for viewing purposes.

Figure 3

Rate constant for flavin oxidation (k _OX) of SidA monitored at 452 nm as a function of pL in the absence or presence of amino acid substrates in H₂O (●) or D₂O (○). (A) Changes in the oxidation rate constant as a function of pH in the absence of amino acid substrate (H₂O₂ elimination). (B) In the presence of Orn (100 mM). (C) In the presence of 15 mM Lys.

Figure 4

Effect of pH on the oxidation of Q102A (●) in the absence of Orn (A) or in the presence of 100 mM Orn (B). In both panels, SidA (π) is shown for comparison.

Figure 5

Active site of SidA showing the FAD (yellow carbons), NADP⁺ (orange carbons), and Orn (cyan carbons) (PDB code 4B63). The figure was made using PyMol.27

Figure 6

Observed conformational changes in SidA upon reduction. The oxidized form is shown with yellow carbons (PDB code 4B63) and the reduced form with white carbons (PDB code 4B65). The only observed changes are the rotation of residues R144 and M101, which are near the N5‐C4a. Typical bending of the FAD in the reduced form is also observed. Rotation of the amide bond is predicted based on the hydrogen bonding interactions with the oxidized flavin (where the amino group acts as a hydrogen bond donor) or reduced flavin (where the carbonyl oxygen acts as a hydrogen bond acceptor).