Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis

Abstract

Butyryl-CoA:acetate CoA transferase, which produces butyrate and acetyl-CoA from butyryl-CoA and acetate, is responsible for the final step of butyrate production in bacteria. This study demonstrates that in the periodontopathogenic bacterium Porphyromonas gingivalis this reaction is not catalyzed by PGN_1171, previously annotated as butyryl-CoA:acetate CoA transferase, but by three distinct CoA transferases, PGN_0725, PGN_1341, and PGN_1888. Gas chromatography/mass spectrometry (GC-MS) and spectrophotometric analyses were performed using crude enzyme extracts from deletion mutant strains and purified recombinant proteins. The experiments revealed that, in the presence of acetate, PGN_0725 preferentially utilized butyryl-CoA rather than propionyl-CoA. By contrast, this preference was reversed in PGN_1888. The only butyryl-CoA:acetate CoA transferase activity was observed in PGN_1341. Double reciprocal plots revealed that all the reactions catalyzed by these enzymes follow a ternary-complex mechanism, in contrast to previously characterized CoA transferases. GC-MS analysis to determine the concentrations of short chain fatty acids (SCFAs) in culture supernatants of P. gingivalis wild type and mutant strains revealed that PGN_0725 and PGN_1888 play a major role in the production of butyrate and propionate, respectively. Interestingly, a triple deletion mutant lacking PGN_0725, PGN_1341, and PGN_1888 produced low levels of SCFAs, suggesting that the microorganism contains CoA transferase(s) in addition to these three enzymes. Growth rates of the mutant strains were mostly slower than that of the wild type, indicating that many carbon compounds produced in the SCFA synthesis appear to be important for the biological activity of this microorganism.

Keywords: CoA transferase; Porphyromonas gingivalis; butyrate; propionate; short chain fatty acid.

FIGURE 1

Proposed metabolic pathways for glutamate and aspartate catabolism in P. gingivalis. This scheme was constructed based on previous reports (Takahashi et al., 2000; Hendrickson et al., 2009). Broken lines indicate predicted pathways that have not been supported by experimental data. Associations of the boxed proteins with the pathways have been determined experimentally (Yoshida et al., 2015, 2016).

FIGURE 2

Initial velocities of crude enzyme extracts of P. gingivalis ATCC 33277 and its mutant strains for butyryl-CoA (A) and propionyl-CoA (B). The reaction mixtures, containing 25 μg/ml crude enzyme extracts, 200 mM sodium acetate, and 1 mM butyryl-CoA or propionyl-CoA, were incubated for 5 min. The concentration of CoA, a reaction byproduct, was measured. Data represent the mean ± standard deviation (n = 3). Asterisks indicate significant differences compared with the wild type (P < 0.01).

FIGURE 3

SDS-PAGE analysis of recombinant P. gingivalis ATCC 33277 proteins. After electrophoresis, samples (∼2 μg) were visualized by Coomassie Brilliant Blue staining. The positions of molecular mass markers (kDa) are shown.

FIGURE 4

GC-MS chromatograms of butyrate and propionate in reaction mixtures. Total ion chromatogram of butyrate (A) and propionate (B) is shown. The mass spectra of each sample agree well with those of the corresponding standard. The reaction mixtures, containing 200 mM sodium acetate and either 1 mM butyryl-CoA or 1 mM propionyl-CoA, with recombinant PGN_0725, PGN_1341, PGN_1888, or PGN_1171 proteins, were incubated for 1 h. After the proteins were removed by acetone precipitation treatment, aqueous phase aliquot (1 μl) was analyzed.

FIGURE 5

Steady-state kinetic analysis of butyryl-CoA:acetate CoA transferase activities of recombinant PGN_0725, PGN_1341, and PGN_1888 proteins. (A) Double reciprocal plots of the initial velocities of acetyl-CoA and butyrate formation from butyryl-CoA and sodium acetate catalyzed by purified recombinant enzymes. Different butyryl-CoA concentrations (0.5–5 mM) were assayed at fixed sodium acetate concentrations (25 mM, filled squares; 50 mM, open squares; 100 mM, filled triangles; 125 mM, open triangles; 200 mM, filled circles; or 250 mM, open circles). (B) Secondary plots of y intercepts (velocity reciprocals) vs. sodium acetate concentrations. (C) Secondary plots of the reciprocal slopes from panel (A) vs. sodium acetate concentration. Data represent the mean ± standard deviation (n = 3).

FIGURE 6

Steady-state kinetic analysis of propionyl-CoA:acetate CoA transferase activities of recombinant PGN_0725 and PGN_1888 proteins. (A) Double reciprocal plots of the initial velocities of acetyl-CoA and propionate formation from propionyl-CoA and sodium acetate catalyzed by purified recombinant enzymes. Different concentrations of propionyl-CoA (0.2–3 mM) were assayed at fixed sodium acetate concentrations (20 mM, filled circles; 25 mM, open circles; 40 mM, filled triangles; 50 mM, open triangles; 75 mM, filled squares; 100 mM, open squares; 125 mM, filled diamonds; 200 mM, open diamonds; and 250 mM, crosses). (B) Secondary plots of y intercepts (velocity reciprocals) vs. sodium acetate concentrations. (C) Secondary plots of the reciprocal slopes from panel (A) vs. sodium acetate concentrations. Data represent the mean ± standard deviation (n = 3).

FIGURE 7

Growth of P. gingivalis strains. P. gingivalis ATCC 33277 (filled circles), PAGU104 (open circles), PAGU108 (filled triangles), PAGU109 (open triangles), PAGU111 (filled squares), PAGU114 (open squares), PAGU115 (filled diamonds), and PAGU118 (open diamonds) were assessed after 6, 12, 24, 36, 48, 60, 72, and 84 h. Representative data for at least two independent experiments are shown.

FIGURE 8

SCFA concentrations in culture supernatants of P. gingivalis ATCC 33277 and mutant strains. The supernatants were obtained by centrifugation of bacterial cultures (OD₆₀₀ 0.375 ± 0.025) and diluted 1:6. Data represent the mean ± standard deviation (n ≥ 3). Asterisks indicate significant differences compared with the wild type (P < 0.01). Diamonds indicate significant differences compared with each no diamond strain (P < 0.01).