Fig. 4: The UcdCFO complex enables anaerobic electron transport from NADH to uroC.
From: Diet-derived urolithin A is produced by a dehydroxylase encoded by human gut Enterocloster species
a Quantification of urolithin concentrations in crude uroC-induced Eb lysates (n = 3 biological replicates) re-treated with DMSO or uroC (350 μM) and various coenzymes at room temperature under anaerobic or aerobic conditions for 20 h. Treatments are indicated by gray squares. NAD(P), nicotinamide adenine dinucleotide (phosphate); FAD, flavin adenine dinucleotide; Na2S2O4, sodium dithionite (reducing agent); HCO2Na, sodium formate (possible electron donor). Data are represented as mean ± SEM; two-way ANOVA with Dunnett’s multiple comparisons test against the Eb lysate incubated with uroC anaerobically (no coenzyme). Statistical significance is denoted in the plot with exact P-values when 0.05 > P ≥ 0.0001 (****). b Volcano plot of untargeted proteomics analysis on DMSO or uroC-treated Eb (n = 3 biological replicates). Data points are colored according to their significance (Fisher’s exact test (two-sided) with Benjamini-Hochberg correction for multiple comparisons). Gray, P-adj ≥ cutoff P-value (0.00048). Blue, P-adj < cutoff P-value (0.00048). c Scatter plot showing the correlation between gene and protein expression (log2FC values) induced in uroC-treated Eb using the datasets in Figs. 2g and 4b, respectively. The non-parametric Spearman rank correlation test (two-sided) was used for statistical analysis, approximate P < 0.0001. d Functional domains in the E. bolteae ucd operon based on InterPro annotations. e LC-MS extracted ion chromatograms (EIC) of uroC ([M-H]-= 243) and uroA ([M-H]- = 227) from a representative anaerobic uroC dehydroxylation assay using crude lysates of R. erythropolis harboring either pTipQC2 (no insert) or pTipQC2-ucdCFO plasmids. Crude lysates were supplemented with 2 mM NADH and incubated anaerobically for 72 h at room temperature. f Quaternary structure prediction of the proteins encoded by the Eb ucd operon using AlphaFold3 (AF3). Small molecule ligands from the X-ray crystal structure of PDB 1RM6 (4-hydroxybenzoyl-CoA reductase from Thauera aromatica) were placed into UcdCFO through structural superposition. Subunits are colored in shades of blue. g X-ray crystal structure of PDB 1RM6 (4-hydroxybenzoyl-CoA reductase from Thauera aromatica). A single αβγ unit (colored in shades of orange) of the (αβγ)2 heterohexamer is shown. h Small molecule ligands from PDB 1RM6 in the superposed UcdCFO model. Source data and statistical details are provided as a Source data file.
