Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Abstract

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guérin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Keywords: Acetyl Coenzyme A; Actinobacteria; Fatty Acid Metabolism; Mass Spectrometry (MS); Mycobacteria; Prokaryotic Signal Transduction; Protein Acylation.

FIGURE 1.

Cyclic AMP-dependent acetylation of FadDs by KATmt. a, two micrograms of purified proteins were incubated in the presence of KATmt (300 ng), cAMP (1 mm), and acetyl CoA (50 μm), followed by Western blotting with acetyl lysine antibody and anti-His antibody. b, CID MS/MS spectrum of the acetylated tryptic peptide ₄₈₃NPTGK^AcILK₄₉₀ of FadD13. The observed fragment ions (b and y ions) are marked on the spectrum and also summarized schematically. c, FadD13_K487A (2 μg) was incubated with KATmt, cAMP, and acetyl CoA, followed by Western blotting with acetyl lysine antibody and anti-His antibody. d, in vitro acetylation of indicated FadDs (500 ng) was performed as indicated, followed by Western blotting with acetyl lysine antibody and anti-His antibody. e, multiple sequence alignment for FadDs that are acetylated by KATmt (upper panel) and those that are not (lower panel). Conserved residues are highlighted by colored boxes. The red box (lower panel) highlights the variation in the highly conserved K/AXP motif. The acetylated lysine is marked with a green dot in both panels. f, dendrogram of full-length sequences of 34 FadDs. The acetylated and non-acetylated FadDs are highlighted with green/red arcs, respectively. Using the same color code and an asterisk, the potential of 12 FadDs to be acetylated by KATmt is predicted.

FIGURE 2.

Deacetylation of FadDs by sirtuin. a, FadD13 (2 μg) was acetylated by KATmt (200 ng) at 37 °C for 30 min and then incubated with sirtuin (500 ng) or sirtuin_H104Y in the presence of NAD⁺ (1 mm) for 1 h at 37 °C. Nicotinamide (NAM, 2 mm) was added to the reaction mixture as indicated. Western blot analysis was performed with acetyl lysine antibody (upper panel) followed by Coomassie Brilliant Blue staining of the blot (lower panel). b, two micrograms of the indicated FadDs were acetylated using KATmt and then deacetylated using sirtuin in the presence of NAD⁺ (1 mm), as described previously, followed by Western blotting with acetyl lysine antibody.

FIGURE 3.

Inactivation of FadDs by KATmt following acylation. a, FadD or acetylated FadD were incubated with ¹⁴C-palmitic acid, ATP (5 mm), and CoA (2 mm), as indicated, at 30 °C for 5 min. Fatty acid migrated on the TLC with a relative mobility of 0.96. Palmitoyl AMP and palmitoyl CoA had Rf values of 0.6 and 0.51, respectively. b, CID MS/MS spectrum of the singly charged species of the propionylated tryptic peptide ₄₈₃NPTGK^PrILK₄₉₀ of FadD13 (m/z 926.4) The signature b and y ions present in the spectrum are marked, and a schematic summary of all the b and y ions obtained is shown. c, FadD13 or propionylated FadD13 were incubated with ¹⁴C-palmitic acid and ATP (5 mm), as indicated, at 30 ^°C for 5 min. d, assays were performed in the presence of varying concentrations of propionyl CoA with a fixed amount of acetyl CoA (50 μm), and acetylated FadD13 was analyzed using Western blot analysis. e, two micrograms of the indicated FadD13 was propionylated using KATmt (200 ng, 30 °C, 30 min) and then deacetylated using sirtuin (500 ng) in the presence of NAD⁺ (1 mm) at 37 °C for 1 h, followed by Western blotting with acetyl lysine antibody.

FIGURE 4.

FadD13 is acetylated by KATmt in a cAMP-dependent manner. a, intracellular cAMP levels during growth of wild-type M. bovis BCG in medium containing 0.2% glycerol or 0.2% glucose as sole carbon source. Data shown are mean ± S.E. of duplicate determinations, with the experiment being repeated thrice. b, whole cell lysates from M. bovis BCG grown in 7H9 media containing 0.2% glycerol or 0.2% glucose were prepared. FadD13 was immunoprecipitated and subjected to Western blot analysis with acetyl lysine antibody and FadD13 antibody. c, semiquantitative RT-PCR analysis of BCG_3114 (FadD13), BCG_1055 (KATbcg), and BCG_1212c (sirtuin) from RNA prepared from wild-type M. bovis BCG grown in 0.2% glucose or 0.2% glycerol as sole carbon source. 16 S primers were used as a normalization control. d, wild-type M. bovis BCG was treated with 0.05% SDS for 1.5 h, and the intracellular cAMP level was measured. All data shown represent the mean ± S.E. of duplicate determinations, with each assay being performed thrice. e, whole cell lysates was prepared from SDS-treated cells. FadD13 was immunoprecipitated and subjected to Western blot analysis with acetyl lysine antibody and FadD13 antibody. f, semiquantitative RT-PCR analysis of FadD13, KATbcg, and sirtuin from RNA prepared from SDS-treated cells. 16 S was used for normalization. g, whole cell lysates from WT, knockout (KO), and complemented (Comp) strains grown in 7H9 media containing 0.2% glycerol or 0.2% glucose were prepared. FadD13 was immunoprecipitated and subjected to Western blot analysis with acetyl lysine antibody and FadD13 antibody.

FIGURE 5.

Propionylation and inactivation of ACS by KATmt. a, CID MS/MS spectrum of the singly charged species of the propionylated tryptic peptide ₆₁₄SGK^PrIMR₆₂₀ of ACS (m/z 747.5). The signature b and y ions present in the spectrum are marked, and a schematic summary of all the b and y ions obtained is shown. b, growth analysis of the WT, knockout (KO), and complemented (Comp) strains in 7H9 media containing 0.2% propionate as the sole carbon source. Shown are the A₆₀₀ values from three independent biological replicates. c, semiquantitative RT-PCR analysis of KATbcg and sirtuin from RNA prepared from wild-type M. bovis BCG grown in 0.2% glucose or 0.2% propionate as the sole carbon source. 16 S primers were used as a normalization control. d, schematic depicting enzymatic inactivation of ACS because of propionylation by KATmt.