Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme

Abstract

Choline and trimethylamine (TMA) are small molecules that play central roles in biological processes throughout all kingdoms of life. These ubiquitous metabolites are linked through a single biochemical transformation, the conversion of choline to TMA by anaerobic microorganisms. This metabolic activity, which contributes to methanogenesis and human disease, has been known for over a century but has eluded genetic and biochemical characterization. We have identified a gene cluster responsible for anaerobic choline degradation within the genome of a sulfate-reducing bacterium and verified its function using both a genetic knockout strategy and heterologous expression in Escherichia coli. Bioinformatics and electron paramagnetic resonance (EPR) spectroscopy revealed the involvement of a C-N bond cleaving glycyl radical enzyme in TMA production, which is unprecedented chemistry for this enzyme family. Our discovery provides the predictive capabilities needed to identify choline utilization clusters in numerous bacterial genomes, underscoring the importance and prevalence of this metabolic activity within the human microbiota and the environment.

Fig. 1.

Discovery of a bacterial gene cluster for anaerobic choline utilization. (A) Generation of trimethylamine (TMA) from choline and its subsequent processing by other organisms. (B) Potential parallel logic between anaerobic choline utilization and bacterial ethanolamine utilization pathways. (C) Putative choline utilization (cut) gene cluster and proposed biochemical pathway for microbial choline metabolism.

Fig. 2.

Bioinformatics identify choline trimethylamine-lyase CutC as a distinct type of glycyl radical enzyme. (A) Maximum likelihood phylogenetic tree showing the relationship between the amino acid sequences of choline TMA-lyases and other biochemically characterized members of the glycyl radical enzyme family. Bootstrap confidence values >50 are indicated on the nodes. (B) Biochemical transformations catalyzed by glycyl radical enzymes.

Fig. 3.

The cut gene cluster is responsible for choline metabolism and TMA production. (A) Growth of D. alaskensis G20 wild-type and C10(pB6) strains at 37 °C on lactate sulfate (black), choline sulfate (red), and choline fermentation (blue) media. The data shown are the average OD₆₀₀ values of four cultures. Error bars represent SEM. (B) LC-MS quantification of d₉-TMA produced by D. alaskensis G20 wild type and C10(pB6) during incubations in (trimethyl-d₉)-choline sulfate and (trimethyl-d₉)-choline fermentation media containing (trimethyl-d₉)-choline chloride (60 mM). Bar graphs represent the mean ± SEM of four cultures. *P < 0.02; **P < 10⁻⁵; ***P < 10⁻⁶. (C) LC-MS quantification of d₉-TMA produced by E. coli BL21(DE3) during incubations in Luria–Bertani (LB) medium containing (trimethyl-d₉)-choline chloride (100 µM). Bar graphs represent the mean ± SEM of four cultures. **P < 10⁻⁵.

Fig. 4.

Growth of D. desulfuricans on choline involves a glycyl radical. EPR spectra of D. desulfuricans ATCC 27774 cell suspensions grown on choline (red) and pyruvate (black) fermentation media. (A) Experimental spectrum of cells grown on choline. (B) Simulation assuming an isotropic signal with g = 2.003, line width (W) = 1.14 mT, and isotropic hyperfine interaction with one proton (A) = 1.3 mT. (C) Experimental spectrum of cells grown on pyruvate. (D) Simulation assuming an isotropic signal with g = 2.004 and line width (W) = 1.94 mT.