Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Abstract

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP⁺ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Keywords: NAD(P)H; ancestral-sequence reconstruction (ASR); crystal structure; enzyme kinetics; flavin adenine dinucleotide (FAD); flavin-containing monooxygenase (FMO); stopped flow.

Figure 1

Catalytic cycle of flavin-containing monooxygenases.A, binding of NADPH and subsequent reduction of the flavin. B, reaction with molecular oxygen and C(4α)-hydroperoxyflavin intermediate formation. C, uncoupling: release of hydrogen peroxide and NADP⁺. D, oxygen atom transfer to a substrate. E, release of water. F, release of NADP⁺.

Figure 2

Ancestral sequence reconstruction of mammalian FMOs. A, Compressed tree of FMOs from jawed vertebrates. The tree depicts the explosion of FMOs in tetrapods. The five FMO clades are shown and those colored in green have been analyzed in this work. B, Close up of the FMO4 and FMO1 clades. Taxonomic distribution is depicted with silhouettes as follows: ancestral tetrapod (), bony fishes (), mammals (), aves () and testudines (). Reconstructed mammalian ancestors are shown with blue circles. On the right the corresponding graphs of the posterior probability distribution per site are shown.

Figure 3

AncFMO1 stopped-flow kinetics.A, reductive half-reaction rates measured in technical triplicates under anaerobic conditions with increasing concentrations of NADH (blue points) or NADPH (red points). The binding constants K_d and k_red values were calculated by fitting the reductive half reaction to the Michaelis–Menten equation with the SD reported in brackets. B, reoxidation spectra of the reduced AncFMO1 with oxygenated buffer over time. C, reoxidation rates with increasing concentration of dioxygen, observed at 368 nm (green triangles) and 448 nm (blue points) in technical triplicates. The dotted lines correspond to the atmospheric concentration of dioxygen (260 μM), present during steady-state kinetics. AncFMO, ancestral flavin-containing monooxygenase; FMO, flavin-containing monooxygenase.

Figure 4

Crystal structure of AncFMO1 and its active site.A, dimeric AncFMO1 with its partially mapped C-terminal helices pointing downward toward the membrane. B, the active site of AncFMO1 in the presence of oxidized coenzyme, NADP⁺, and two glycerol molecules (green) is depicted, with the key residues labeled. C, extensive hydrogen bond interactions between E281, N61, V59, FAD, NADP⁺, and a glycerol molecule are shown to illustrate key intermolecular interactions and potential substrate binding modes, represented by dashed yellow lines. FAD, NADP⁺, and DDM molecules are colored in yellow, cornflower blue, and white, respectively. AncFMO, ancestral flavin-containing monooxygenase; DDM, dodecyl-β-D-maltoside; FAD, flavin adenine dinucleotide.

Figure 5

Unique structural features of AncFMO1.A, the differing 2’-phosphate binding site of NADP⁺ for AncFMO1 (left, side chains in cyan) compared with AncFMO3-6 (PDB:6SE3) (right, side chains in dark purple) is shown with key hydrogen-bonding interactions shown as yellow dashed lines. NADP⁺ is shown in cornflower blue. B, the conformation adopted by residues 416 to 425 that reaches out toward the α-helical triad in a large arched conformation is indicated by the black arrow. Super positioning AncFMO1 (red) against AncFMO3-6 (dark purple) conveys the new structural topology. C, the large active site cavity (approximately 19 Å wide) is depicted in magenta and stretches out toward the solvent and the membrane–protein interface (indicated with a black arrow). The side chain of E281 is shown in green pointing toward the isoalloxazine ring and the active site. FAD, NADP⁺, and glycerol molecules are shown in yellow, cornflower blue, and green, respectively. AncFMO, ancestral flavin-containing monooxygenase; FAD, flavin adenine dinucleotide.