Gut bacterium Blautia sp. MRG-PMF1 cleaves bulkier isoflavone ether bonds

A cobalamin-dependent O-demethylase carried by the human gut bacterium Blautia sp. MRG-PMF1 has now been shown to strip alkyl groups larger than methyl from isoflavone derivatives, a capability that had not previously been demonstrated for this class of enzyme. The finding, published in Applied Biological Chemistry, extends the known reactivity spectrum of corrinoid-dependent O-demethylases from O-demethylation and O-deallylation to broader O-dealkylation, and it suggests that the human gut microbiota may process a wider range of dietary natural products and xenobiotics than assumed.

Blautia sp. MRG-PMF1 was previously isolated for its role in biotransforming polymethoxyflavones in the human gut. Its O-demethylase system is a cobalamin, or vitamin B12, dependent methyltransferase machinery that cleaves C–O bonds in aryl methyl ether groups through a Co(I) corrinoid-mediated SN2 mechanism. The system comprises four components: methyltransferase I, which binds the aryl methyl ether substrate and activates the otherwise inert ether linkage by Zn2+ coordination; a corrinoid protein whose Co(I) nucleophile attacks the methyl carbon to form methyl-Co(III)balamin; methyltransferase II, which transfers the methyl group from the methylated corrinoid protein to tetrahydrofolate, generating N5-methyltetrahydrofolate; and an activating enzyme that maintains the corrinoid cofactor in its catalytically active Co(I) oxidation state, reversing inadvertent oxidation to Co(II) with ATP hydrolysis and electrons from donors such as ferredoxin.

Recent work had already revealed unusual substrate flexibility in this machinery. The bacterium can cleave not only conventional methyl aryl ether linkages but also allyl aryl ether bonds through what appears to be an SN2′-type nucleophilic conjugated substitution mechanism, a reactivity with implications for the metabolism of polymethoxyflavones, curcuminoids, icariin and furanocoumarins. That evidence challenged the prevailing assumption that the biological activity of this enzyme is strictly limited to methyl group procurement from aryl methyl ethers. Encouraged by this flexibility, the researchers set out to determine whether the catalytic activity could extend beyond methyl aryl ethers to structurally related alkyl ether-containing isoflavones.

To probe the enzyme’s reactivity, the team selected four model substrates: 7-methoxyisoflavone, 7-ethoxyisoflavone, 7-propoxyisoflavone and 7-isopropoxyisoflavone, the last of which is the synthetic flavonoid drug ipriflavone, clinically utilized in the treatment of osteoporosis. The 7-propoxy and 7-ethoxy compounds were synthesized in the laboratory by reacting 7-hydroxyisoflavone with the corresponding alkyl iodides in the presence of potassium carbonate in anhydrous dimethylformamide, while the methoxy derivative and ipriflavone were purchased commercially. Whole-cell biotransformations were carried out under strict anaerobic conditions of 5% CO2, 10% H2 and 85% N2 at 35 °C, with cultures sampled at intervals between 0 and 96 hours and analyzed by high-performance liquid chromatography with diode array detection.

The whole-cell experiments showed that all four 7-alkoxyisoflavones were converted to 7-hydroxyisoflavone. 7-Methoxyisoflavone, which eluted at 25.8 minutes on the HPLC column, was successfully converted to 7-hydroxyisoflavone, eluting at 17.7 minutes, as confirmed by comparison with an authentic reference standard. An unknown compound detected at 14.7 minutes was structurally identified by LC–MS and determined to be a systematic impurity unrelated to the dealkylation pathway. The biotransformation of 7-ethoxyisoflavone proceeded slowly during the first 3 hours, but its conversion efficiency after 24 hours was comparable to that of the methoxy substrate. 7-Propoxyisoflavone exhibited a time-dependent conversion profile similar to that of 7-methoxyisoflavone over the 24-hour incubation, while 7-isopropoxyisoflavone, which eluted at 33.4 minutes, was converted to 7-hydroxyisoflavone to a lesser extent than the methyl and primary alkyl ether compounds. Nevertheless, the results confirm that Blautia sp. MRG-PMF1 can metabolize ipriflavone in vitro.

To elucidate the reaction mechanism of this newly identified cobalamin-dependent O-dealkylase, the researchers assessed O-dealkylation activity using cell-free extracts prepared from 4 liters of anaerobic culture. Cell pellets were washed with degassed anaerobic phosphate buffer, lysed by sonication under argon with sodium dithionite added, and the supernatant was kept in an anaerobic chamber to prevent oxygen exposure. Substrate solutions were added to the cell-free extract, which had a protein concentration of 4.59 ± 0.12 mg/ml, and incubated for 60 minutes under strictly anaerobic conditions, with all reactions performed in duplicate. Conversion was quantified by monitoring the decrease in substrate concentration.

The cell-free extract reactions revealed distinct substrate conversion efficiencies after 1 hour of incubation. 7-Ethoxyisoflavone was converted fastest, at 77.62 ± 0.43%, followed by 7-methoxyisoflavone at 70.21 ± 0.37% and 7-propoxyisoflavone at 64.82 ± 0.35%, while 7-isopropoxyisoflavone exhibited the lowest reactivity at 32.34 ± 0.87%. The observed reactivity followed the order of O-ethyl > O-methyl > O-propyl > O-isopropyl, indicating that primary alkyl aryl ethers are more readily dealkylated than secondary ethers, consistent with the trends observed in the whole-cell biotransformation. Although the reaction data were not accompanied by kinetic data from purified enzymes, which remains a challenge for this multi-component system, it was clear that the O-demethylase in Blautia sp. MRG-PMF1 catalyzes the cleavage of alkyl aryl ether bonds.

The reactivity order carries mechanistic weight. If a radical mechanism prevailed, a faster conversion of 7-isopropoxyisoflavone relative to the other 7-alkoxyisoflavones would have been expected, owing to the greater stability of its secondary radical intermediate. Instead, the observed order strongly suggests a polar SN2-type mechanism in which the Co(I) center of the corrinoid protein attacks the alkyl carbon of the Zn2+-polarized ether bond, generating an alkylcob(III)alamin adduct and the demethylated aromatic product. The slightly faster cleavage of the ethyl ether compared with the methyl ether can be explained by the greater polarization of its C–O bond, which could have enhanced reactivity enough to override the steric hindrance presented by the ethyl group during the Co(I) nucleophilic attack. For the methyl adduct, the electrophilic methyl group is transferred to tetrahydrofolate bound at methyltransferase II; for the other alkyl adducts, homolytic cleavage of the Co(III)–C bond could generate alkyl radical species and Co(II) corrinoid protein.

The significance of the finding reaches beyond enzymology. The authors note that the gut metabolism of pharmaceuticals such as ipriflavone, as well as agrochemicals like flutolanil, might be subject to transformation by this enzyme, highlighting the need to reconsider how corrinoid-dependent O-demethylases contribute not only to dietary phytochemical bioactivation but also to the metabolic fate of xenobiotics in the human gut. Enzymes of this type evolved primarily to enable anaerobic bacteria to utilize methylated aromatics as sources of carbon and energy, playing a central role in the biodegradation of plant-derived lignin components and other aromatic xenobiotics, and their activity initiates one-carbon metabolism within the bacterial cell while producing metabolites with potentially enhanced bioactivity in the human body.

Mechanistically, the finding also sits alongside recent work showing that Blautia sp. MRG-PMF1 can catalyze deprenylation of furanocoumarins, cleaving prenyl aryl ether bonds to convert imperatorin and isoimperatorin into their deprenylated products, and that the enzyme can adopt an SN2′ mechanism distinct from the classical SN2 pathway governing aryl methyl ether cleavage. With the present study, the same corrinoid-dependent machinery is now implicated in O-dealkylation of ethyl, propyl and isopropyl substituents, providing the first definitive evidence of this activity in Blautia sp. MRG-PMF1 and expanding the known reactivity spectrum of cobalamin-dependent O-demethylases. The work was performed with in vitro whole-cell and cell-free extract assays of a single bacterial strain, and the authors note that kinetic characterization with purified enzymes remains an open challenge, leaving the precise physiological contribution of this O-dealkylation activity within the complex human gut community to be determined.

Subject of Research: Cobalamin-dependent O-dealkylation of isoflavone derivatives by the human gut bacterium Blautia sp. MRG-PMF1

Article Title: Gut microbial O-dealkylation of isoflavone derivatives by Blautia sp. MRG-PMF1

Article References: Chaiyasarn, S., Khumthong, I., & Han, J. (2026). Gut microbial O-dealkylation of isoflavone derivatives by Blautia sp. MRG-PMF1. Applied Biological Chemistry, 69(1), Article 56. https://doi.org/10.1186/s13765-026-01122-0

Image Credits: AI Generated

DOI: 10.1186/s13765-026-01122-0

Keywords: Blautia sp. MRG-PMF1, isoflavones, O-dealkylation, cobalamin-dependent O-demethylase, gut microbiota, 7-hydroxyisoflavone, ipriflavone, SN2 mechanism, corrinoid protein, polymethoxyflavones, xenobiotic metabolism, anaerobic bacteria