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.
Highlights
For dealing with endogenous and foreign toxic compounds, mammals and other animals developed oxidative systems that clear such potentially harmful elements from cells and tissues [1]
Ancestral flavin-containing monooxygenase (AncFMO)1 was reconstructed with high confidence
We characterized in depth the ancestors of hFMO2, hFMO3, and hFMO5
Summary
Sequence analysis has shown that the FMO paralogs emerged at the time of tetrapod evolution and all five FMO paralogs were encoded in the genome of the mammalian ancestor, 177 mya [24] (Fig. 2, Fig. S1, Table S1). The purified enzyme was incubated for 1 h at 30 C with indole and a NADPH regeneration system, which resulted in formation of indigo (Fig. S4) These data demonstrated that AncFMO1 featured all the typical properties of FMOs and was able to convert known FMO1 substrates. With Leu150 not being able to create hydrogen bonds, Lys373 is not tied down and can extend outward, toward a glycerol molecule situated below the diphosphate moiety of the NADP+, thereby further enlarging the active site (Fig. 4B) With these residues being conserved between AncFMO1 and hFMO1, it is likely that these features would be observed for hFMO1, further validating the use of AncFMO1 as a structural model. Refinement R-work (%) R-free (%) Number of non–hydrogen atomsc RMS (bonds) Å RMS (angles) Ramachandran favored (%) Ramachandran allowed (%) Ramachandran outliers (%) Average B-factor
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