The dynamics of the flavin, NADPH, and active site loops determine the mechanism of activation of class B flavin-dependent monooxygenases

Abstract

Flavin-dependent monooxygenases (FMOs) constitute a diverse enzyme family that catalyzes crucial hydroxylation, epoxidation, and Baeyer-Villiger reactions across various metabolic pathways in all domains of life. Due to the intricate nature of this enzyme family's mechanisms, some aspects of their functioning remain unknown. Here, we present the results of molecular dynamics computations, supplemented by a bioinformatics analysis, that clarify the early stages of their catalytic cycle. We have elucidated the intricate binding mechanism of NADPH and L-Orn to a class B monooxygenase, the ornithine hydroxylase from $\text{Aspergillus}$ $\text{fumigatus}$ known as SidA. Our investigation involved a comprehensive characterization of the conformational changes associated with the FAD (Flavin Adenine Dinucleotide) cofactor, transitioning from the out to the in position. Furthermore, we explored the rotational dynamics of the nicotinamide ring of NADPH, shedding light on its role in facilitating FAD reduction, supported by experimental evidence. Finally, we also analyzed the extent of conservation of two Tyr-loops that play critical roles in the process.

Keywords: NADPH binding; NADPH dynamics; flavin dynamics; flavin-dependent monooxygenases; ornithine binding; uncoupling.

FIGURE 1

Scheme of the putative events occurring at the initial stages in the catalytic mechanism of SidA. The computational models appearing in the figure, except for those enclosed in a rectangle, were prepared from PDB files as explained in the main text. All the events indicated with a green arrow were studied via US simulations. Those highlighted with a thick green line correspond to the most probable pathway according to the results of this work.

FIGURE 2

PMF for the binding of NADPH and L‐Orn to the resting form of SidA (SidA(${\mathrm{FAD}}_{\text{out}}$)). The calculations we carried out to assess their consistency and the procedure we followed to compute the error bars are presented in the Supporting Information section.

FIGURE 3

Representative structures observed during the binding of NADPH to SidA(${\mathrm{FAD}}_{\text{out}}$). NADPH is highlighted in yellow. Structures were selected by visual inspection. The values of the RC at which the snapshots were taken are shown below each panel. Residues that bear significant interactions with NADPH are represented in $\mathit{\text{licorice}}$. Panel (a): NADPH interacts via its phosphate groups with positive residues (Arg364, Lys379, and Lys393) located at the border of the enzyme. Panel (b): The adenine moiety of NADPH reaches its final position and interacts with Lys361, Arg279, and Ser218. Panel (c): Two alternative snapshots that illustrate the displacement of the Tyr407‐loop as the nicotinamide ring of NADPH gets close to the FAD cofactor. Panel (d): The nicotinamide ring of NADPH is sandwiched between the si‐face of FAD and the phenyl group of Tyr324. Panel (e): At the end of the process, Tyr324 is displaced out and NADPH reaches its final conformation.

FIGURE 4

Representative conformations observed during the binding of L‐Orn to SidA(${\mathrm{FAD}}_{\text{out}}$). L‐Orn is highlighted in yellow. Structures were selected by visual inspection. The values of the reaction coordinate at which the snapshots were taken are shown below each panel. Residues Arg279 and Asp280, together with Tyr324 and ${\mathrm{FAD}}_{\text{out}}$ are represented in $\mathit{\text{licorice}}$. Panel (a) describes the initial interaction between L‐Orn and Arg279/Asp280. Panel (b) represents a conformation in which L‐Orn is entering the active site. It is flanked by the phenyl ring of Tyr324, on one side, and by the si‐face of ${\mathrm{FAD}}_{\text{out}}$ on the other. Panel (c): L‐Orn docked in its active site pocket.

FIGURE 5

PMFs for the out to in transition of the FAD cofactor. The calculations we carried out to assess their consistency and the procedure we followed to compute the error bars are presented in the Supporting Information section. The red curve represents the profile corresponding to the SidA(${\mathrm{FAD}}_{\text{out}}$, NADPH) $\to$ SidA(${\mathrm{FAD}}_{\text{in}}$, NADPH) transition, while the blue one indicates the SidA(${\mathrm{FAD}}_{\text{out}}$, NADPH, L‐Orn) $\to$ SidA(${\mathrm{FAD}}_{\text{in}}$, NADPH, L‐Orn) transition.

FIGURE 6

Probability density function for the distances between the H‐atoms bound to C4(NADPH) and N5(FAD). The blue and light‐blue distributions were computed before the rotation of NADPH around the C1D‐N1N bond, while the red and orange functions were obtained after the rotation.

FIGURE 7

Panels (a and b) illustrate the active site of SidA(${\mathrm{FAD}}_{\text{in}}$, NADPH) with the nicotinamide moiety of NADPH before and after rotation, respectively. Tyr324, Tyr407, FAD, and NADPH are drawn as $\mathit{\text{licorice}}$. The two Tyrosine residues are shadowed. The pro‐R hydrogen atom is highlighted with a blue sphere. Panel (c) shows the PMFs for the rotation of the nicotinamide ring of NADPH in models SidA(${\mathrm{FAD}}_{\text{in}}$, NADPH) (blue line) and SidA(${\mathrm{FAD}}_{\text{in}}$, NADPH, L‐Orn) (red line). The calculations we carried out to assess the consistency of their and the procedure we followed to compute the error bars are presented in the Supporting information section.

FIGURE 8

Sequence logos built using data derived from class‐B FMOs and SidA homologs obtained from BlastP and Jackhmmer. The number of sequences for each case is 24 (class‐B), 173 (BlastP), and 437 (Jackhmmer). The percentage of conservation of the sequence motifs considered in each case is indicated below. FMO, flavin‐dependent monooxygenase.

FIGURE 9

Relevance of SidA residues according to Consurf (panel a) and the Evolutionary Trace algorithms (panel b). Both Tyr324 and Tyr407 loops are circled in both panels. Arg279 is underlined in green. As ET server depicts the results on the structure residues, regions with missing residues are indicated with an asterisk. These are at the beginning of the sequence, in positions 379–383, and at the end.