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.
Highlights
Periodontal diseases are a group of inflammatory conditions that lead to the destruction of tooth-supporting tissues and appear to be associated with serious systemic conditions (Kumar, 2013)
PGN_1171 has been previously annotated as a butyryl-CoA:acetate CoA transferase of P. gingivalis ATCC 33277 (Nelson et al, 2003; Hendrickson et al, 2009), the deduced PGN_1171 amino acid sequence shared no more than 20 and 27% identity with R. hominis RHOM_13820 protein and Clostridium aminobutyricum AbfT protein, respectively, both of which were experimentally shown to function as butyryl-CoA:acetate CoA transferases (Scherf and Buckel, 1991; Charrier et al, 2006)
A database search revealed greater RHOM_13820 amino acid identity with PGN_0725 (38.5%), PGN_1341 (23.7%), and PGN_1888 (36.4%) proteins, all of which have been annotated as putative CoA transferases in P. gingivalis ATCC 33277 (Naito et al, 2008), than PGN_1171
Summary
Periodontal diseases are a group of inflammatory conditions that lead to the destruction of tooth-supporting tissues and appear to be associated with serious systemic conditions (Kumar, 2013). P. gingivalis releases large amounts of butyrate and propionate into its culture medium (Niederman et al, 1996; Imai et al, 2012) These molecules penetrate the periodontal tissue because of their low molecular weights (Tonetti et al, 1987) and subsequently disturb host cell activity and host defense systems (Singer and Buckner, 1981; Eftimiadi et al, 1990; Kurita-Ochiai et al, 1995). Concentrations of these molecules in the periodontal pockets significantly correlate with the clinical measure of disease severity and inflammation (Niederman et al, 1997; Qiqiang et al, 2012). Butyrate produced by bacteria is thought to play an important and beneficial role (Siavoshian et al, 2000; Peng et al, 2009; Plöger et al, 2012; Qin et al, 2012; Le Chatelier et al, 2013; Mathewson et al, 2016)
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