Identification of an Inhibitor of Microbial TMA Formation from Choline

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Balskus E.P. Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme. Figure S1 Characterization of Choline TMA Lyase Activity of P. mirabilis and Recombinant CutC/D from P. mirabilis and D. alaskensis, Related to Figure 1 Show full caption (A) E. coli Top10 cells were transformed with empty pBAD vector (negative control), pBAD-cutC/D (from P. mirabilis), or the indicated catalytically inactive cutC point mutants, as described in Experimental Procedures . Cells were grown, CutC/D expression was induced with 0.2% arabinose, and then cells collected by centrifugation, re-suspended in 2x culture volumes of PBS and incubated at 37°C in gas tight reaction vials with 100 μM of d9-choline substrate. At the indicated time points, TMA lyase activity was measured as d9-TMA production using stable isotope dilution LC/MS/MS. (B) Effect of the indicated choline analogs on choline TMA lyase activity of P. mirabilis lysate. Protein lysate (3 mg) was incubated with d9-choline (100 μM) in 2 ml PBS with the indicated concentrations of choline analogs (P-choline, betaine, TMSi-ETOH, and DMB) at 37°C for 23 hr in gas tight reaction vials and choline TMA lyase activity measured as the production of d9-TMA, as described under Experimental Procedures 600 nm = 1.5), were pelleted, re-suspended in 6x original culture volume of cold argon-sparged PBS containing d9-choline (100 μM) substrate ± DMB (2 mM) and incubated in gas-tight reaction vials under argon blanket at 37°C for 12 hr. Choline TMA lyase activity was quantified by the production of d9-TMA by stable isotope dilution LC/MS/MS. (C) E. coli Top10 cells were transformed with pUC57 plasmid (negative control) or pUC57 expression vectors constitutively expressing D. alaskensis cutC alone, cutD alone, cutC- intergenic region-cutD, or the indicated catalytically inactive cutC point mutants, as described in Experimental Procedures . Bacterial cells at stationary phase (OD= 1.5), were pelleted, re-suspended in 6x original culture volume of cold argon-sparged PBS containing d9-choline (100 μM) substrate ± DMB (2 mM) and incubated in gas-tight reaction vials under argon blanket at 37°C for 12 hr. Choline TMA lyase activity was quantified by the production of d9-TMA by stable isotope dilution LC/MS/MS. Data represent mean ± SE from three independent replicates (A-C). NA, no addition. Anaerobic choline degradation is believed to serve as the major source of TMA formation within the intestines (). Therefore, we focused initial drug screening efforts on the identification of potential inhibitors of microbial TMA production from choline- or so-called choline-TMA lyase activity. The cleavage of the C-N bond of choline to form TMA and acetaldehyde was reported with extracts of Proteus mirabilis, a known human commensal microorganism that is easily cultured, nearly 3 decades ago (). Therefore, we initially used P. mirabilis in early drug screening studies. However, during the course of our drug discovery efforts, the identification of a microbial choline TMA lyase was reported as a unique glycyl radical enzyme complex composed of a catalytic polypeptide, CutC, and an associated activating protein, CutD, encoded by adjacent genes within a gene cluster (). After this report, we subsequently cloned and expressed the cutC/D genes from P. mirabilis and confirmed that the expressed cutC/D gene complex serves as a microbial choline TMA lyase enzyme source ( Figure S1 A). For example, intact E. coli Top10 cells in culture lack choline TMA lyase activity, but following transformation with both P. mirabilis cutC/D genes, they acquire the capacity to enzymatically cleave choline to produce TMA. To further confirm that microbial TMA lyase enzyme activity monitored with this system required the presence of both CutC and CutD, we examined multiple additional controls, including the demonstration of the lack of choline TMA lyase activity following transformation with only cutC or cutD individually or following transformation with cutD but in combination with point mutants of cutC previously reported () to lack TMA lyase activity (cutC C781A or G1117A of P. mirabilis were made, which correspond to mutation of the conserved thiyl and glycyl-radical active-site residues C489 and G821 in D. alaskensis) ( Figure S1 A).

Wang et al., 2014b Wang Z.

Tang W.H.

Buffa J.A.

Fu X.

Britt E.B.

Koeth R.A.

Levison B.S.

Fan Y.

Wu Y.

Hazen S.L. Prognostic value of choline and betaine depends on intestinal microbiota-generated metabolite trimethylamine-N-oxide. 50 ) in the ∼10 μM range in the recombinant cutC/D system ( Figure 1 The Choline Analog DMB Inhibits Microbial Choline TMA Lyase Activity Show full caption (A) Effect of the indicated choline analogs on microbial TMA lyase activity (measured as d9-TMA production from 100 μM of the indicated d9-labeled substrate) from the lysate of E. coli Top10 cells transformed (pBAD vector) with cutC/D genes (from P. mirabilis). (B) Effect of DMB on choline TMA lyase activity in intact P. mirabilis incubated with the indicated concentrations of d9-choline substrate with or without DMB (1 mM) at 37°C. NA, no addition. 600 nm = 0.5) cells were pelleted and then re-suspended in minimal media supplemented with the indicated concentrations of d9-choline with or without DMB (2 mM) for 15 min at 37°C. Intracellular d9-choline was then quantified as described in the (C) DMB effect on choline uptake. P. mirabilis (OD= 0.5) cells were pelleted and then re-suspended in minimal media supplemented with the indicated concentrations of d9-choline with or without DMB (2 mM) for 15 min at 37°C. Intracellular d9-choline was then quantified as described in the Experimental Procedures (D) Choline TMA lyase activity from intact E. coli Top10 cells transformed with the indicated constructs in the presence versus absence of DMB. (E) DMB dose-response curves for inhibition of choline TMA lyase activity in intact E. coli Top10 cells transformed with cutC/D genes from either D. alaskensis (pUC57 vector) or P. mirabilis (pBAD vector). (F) TMA lyase activity for the indicated substrates in P. mirabilis lysate with or without DMB. Data are presented as mean ± SE from three independent replicates. See also Figures S1 S2 , and S6 We surveyed multiple different natural and synthetic structural analogs of choline for their effects on the two microbial choline TMA-lyase activity systems (microbial TMA production using P. mirabilis lysate or recombinant choline TMA lyase activity using cutC/D from P. mirabilis transformed into E. coli) and noted three distinct patterns. Similar results were observed with both systems, with data shown in Figure 1 A being from cutC/D-transformed E. coli cell lysates and illustrative data from P. mirabilis lysates shown in Figure S1 B. The majority of choline analogs showed no effect on TMA lyase activity, which was monitored by quantifying the impact of the analogs on d9-TMA formation from a fixed amount of [d9-N,N,N-trimethyl]choline (d9-choline) substrate. One notable example in this category is betaine ( Figure 1 A; Figure S1 B), a choline oxidation metabolite and food component that, we recently showed, can serve as a substrate for gut-microbe-dependent TMA and TMAO generation in vivo, albeit at a rate approximately 100-fold less than that observed with a comparable amount of ingested choline (). Two of the choline analogs examined, 3,3-dimethyl-1-butanol (DMB) and 2-(trimethylsilyl)ethanol (TMSi-ETOH), in which the nitrogen in choline is replaced with either a carbon or silicon atom, respectively, showed roughly comparable cutC/D choline TMA lyase inhibitory activity with a half-maximal inhibitory concentration (IC) in the ∼10 μM range in the recombinant cutC/D system ( Figure 1 A). Interestingly, the phosphonium analog of choline (P-choline) showed a unique effect by augmenting cutC/D choline TMA lyase activity ( Figure 1 A; Figure S1 B), suggesting that this choline analog acts as an agonist and may promote more effective interaction between the catalytic (CutC) and activating (CutD) polypeptides. Of the choline TMA lyase activity inhibitors identified, we suspected that DMB, given its structure, might be relatively non-toxic, and possibly even found as a natural product in existing foods or alcoholic beverages. Indeed, screening of multiple fermented liquids, oils, and distilled products confirmed that it is present in some alcoholic beverages and oils to varying levels (e.g., DMB was detected in some balsamic vinegars, in red wines, and in some cold-pressed extra virgin olive oils and grapeseed oils; highest levels observed were 25 mM; data not shown). Therefore, we elected to take DMB forward as a potential tool drug with which to test the hypothesis that inhibiting microbial production of TMA might serve as a potential therapeutic approach for the treatment of atherosclerosis.