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Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs

A Publisher Correction to this article was published on 21 January 2020

This article has been updated

Abstract

Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate–histidine switch is speculated to induce the distinctive Baeyer–Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.

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Fig. 1: The catalytic mechanism of FMOs.
Fig. 2: Ancestral-sequence reconstruction of FMOs.
Fig. 3: Stopped-flow kinetics studies on AncFMO2 and AncFMO3-6.
Fig. 4: Crystal structures of the AncFMOs.
Fig. 5: Active sites of the AncFMOs.
Fig. 6: Substrate tunnels and structural differences in the AncFMOs.

Data availability

Coordinates and structure factors have been deposited with the Protein Data Bank with accession codes 6SEM (AncFMO2), 6SF0 (AncFMO2 in complex with NADP+), 6SE3 (AncFMO3-6), 6SEK (AncFMO5). Source data for Figs. 2 and 3, and Table 1, are available with the paper online.

Change history

  • 21 January 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

The research for this work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 722390; the Italian Ministry of Education, University and Research (MIUR) under the “Dipartimenti di Eccellenza (2018–2022)” program; and ANPCyT (Argentina) PICT 2016-2839 to M.L.M. M.L.M. is a member of the Research Career of CONICET, Argentina. The authors thank M. J. Ayub for his valuable comments and contributions on the manuscript.

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Contributions

All listed authors performed experiments and analyzed data. C.R.N. generated purification protocols, crystallized the AncFMOs, collected the corresponding datasets at the ESRF and SLS facilities, performed structural analysis and elucidated the AncFMO structures. G.B. and C.R.N. performed Golden Gate cloning to insert the AncFMO genes into their respective vectors, designed by G.B. G.B., C.R.N. and F.F. carried out mutagenesis and extensive kinetic analysis and validated the substrate profiles using stopped-flow ultraviolet–visible spectroscopy and GCMS for each AncFMO. M.L.M. conducted thorough evolutionary analyses and performed ancestral-sequence reconstruction to obtain AncFMO protein sequences. C.R.N., G.B. and M.L.M. prepared the figures. C.R.N. wrote the manuscript and A.M., M.W.F. and M.L.M. edited it. All authors provided critical feedback and helped shape the research, analysis and manuscript. A.M. and M.W.F. conceived the original idea.

Corresponding authors

Correspondence to María Laura Mascotti or Marco W. Fraaije or Andrea Mattevi.

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The authors declare no competing interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Crystal Structure of AncFMO2 in the absence of NADP+ (green) superposed to the structure bound to NADP+ (dark green).

AncFMO2 crystallizes in an identical manner with or without NADP+. The root-mean-square deviation between the native AncFMO2 and its NADP+ complex is 0.23 Å over 530 Cα atom pairs. The orientation of the dimer depicts the structure sitting on top of the phospholipid bilayer as shown in the other structures (see Fig. 4).

Extended Data Fig. 2 Electron density maps of the AncFMOs.

Structure determination was greatly facilitated by density averaging because the asymmetric unit of the crystals of AncFMO2, AncFMO3-6 and AncFMO5 contain four, six and two protein molecules, respectively. The depicted 2Fo-Fc maps were calculated by averaging the electron density maps obtained after molecular replacement (shown in blue). a, AncFMO2 shown in lime green, without NADP.+ b, AncFMO2 shown in lime green, with NADP.+ c, AncFMO3-6 shown in dark magenta, with NADP.+ d, AncFMO5 shown in dark orange, with NADP.+ Cofactors FAD and NADP+ are shown in yellow and cornflower blue respectively. The contour level is 1.4 σ.

Extended Data Fig. 3 The crystal packing of AncFMO2 forms multiple planes of soluble dimer–dimer interactions that extend across the lattice.

The asymmetric unit is depicted by the four differently colored monomer units of dark yellow, dark red, dark green and dark blue. In between each plane, we see multiple transmembrane helices projecting upwards and downwards from each asymmetric unit. Each dimer projects its transmembrane helices towards its reciprocal dimer.

Extended Data Fig. 4 Topological features of the mammalian FMOs.

a, Highly conserved NADP(H) and FAD dinucleotide-binding domains that are observed in all FMOs. b, The characteristic 80-residue insertion (residues 214–295 in AncFMO3-6) that covers the FAD and binds to the membrane monotopically through an α-helical triad. c, The additional C-terminal (residues 443–528) that orchestrates both monotopic and bitopic membrane binding features through an α-helical triad and a C-terminal helix respectively.

Extended Data Fig. 5 NADPH oxidase activity and melting temperature of human FMO3.

a, NADPH consumption was not altered by the presence of substrate and so a Michaelis–Menten curve was plotted at differing NADPH concentrations. The KM and kcat for NADPH were determined at 46 ± 9 µM and 0.06 ± 0.16 s–1, respectively. b, Extensive buffer screenings for human FMO3 resulted in a maximum melting temperature of 44.5 °C (with and without 200 μM NADP+ in the upper and lower curves, respectively) in buffer conditions of 100 mM HEPES pH 7.5, 10 mM KCl and 0.05% (v/v) TRX-100-R. All measurements were performed in technical duplicates.

Extended Data Fig. 6 Differing residues between AncFMOs and human FMOs.

The upper and lower panels display the structures in two orientations. The changes exhibited by AncFMO2, AncFMO3-6 and AncFMO5 compared to human FMOs are shown in lime green, dark magenta and orange, respectively. A close systematic analysis of the changes does not reveal any clear pattern of amino acid substitutions. Most are localized in the membrane-binding regions, implying that the enzyme can undergo multiple mutations in these parts of the protein as long as the hydrophobic nature of the side chains is conserved. This finding is further corroborated by the sequence alignment of the human FMOs and the AncFMOs sequences, with the sequences at the subdomains and C-termini varying substantially (Supplementary Fig. 4). The mutations, however, are bereft in the FAD and NADP(H) binding domains, describing well-conserved sequence motifs among FMOs. Furthermore, the residues inside the enzyme and more importantly, lining the tunnels, are also well conserved. Only one overwhelming change in the core of the enzyme is observed in AncFMO5, as shown in Fig. 6d (lower panel).

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Nicoll, C.R., Bailleul, G., Fiorentini, F. et al. Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs. Nat Struct Mol Biol 27, 14–24 (2020). https://doi.org/10.1038/s41594-019-0347-2

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