Reversible acetylation and inactivation of Mycobacterium tuberculosis acetyl-CoA synthetase is dependent on cAMP.

Protein acetylation was first discovered in histones almost 50 years ago (1). In the following four decades, it became clear that the histone acetyltransferases (HAT’s) were part of a very large enzyme superfamily referred to as the GCN5-related N-acetyltransferases (GNAT’s). The GNAT superfamily catalyzes acetyl transfer from acetyl coenzyme A (AcCoA) to a primary amine within a small molecule or a protein and is one of the largest superfamilies with over 30,000 members in all kingdoms of life (2). However, the biological functions and/or substrates for a majority of the GNAT’s are largely unknown, with only 5 of the 26 GNAT’s in Escherichia coli having known biological functions. But it is clear that some of the pro- and eukaryotic GNAT’s must be capable of acetylating proteins other than histones. In the last five years, several research groups have identified over 2,000 proteins acetylated at the e-amino group of a lysine residue in human cells (3–5), and almost 200 in E. coli and Salmonella enterica (6–8). In addition to protein phosphorylation, protein lysine acetylation is now recognized as a ubiquitous and evolutionarily conserved protein modification from prokaryotes to eukaryotes. Both of these post-translational modifications are known to be reversible, with protein Ser/Thr and Tyr phosphatases and protein deacetylases (sirtuins) found in all organisms containing the corresponding kinases and acetyltransferases. In the case of protein acetylation, many of the enzymes involved in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle and fatty acid metabolism were found to be acetylated, implying an extensive role of acetylation in the regulation of intracellular metabolism (3–8). Despite the remarkable number of proteomically identified acetylated proteins, the effect of acetylation on the activity of most of the “identified” enzymes, or the particular GNAT responsible for acetylation, remains largely unknown. In Mycobacterium tuberculosis, the causative agent of tuberculosis, only five out of twenty predicted GNAT’s have been biochemically, functionally or structurally characterized (9–14). Two other enzymes, Rv0995 (RimJ) and Rv3420c (RimI) are predicted to acetylate the N-terminus of ribosomal proteins S5 and S18, respectively (2). Of the twenty GNAT’s, Rv0998 exhibits a unique structural feature with a C-terminal GNAT domain fused to an N-terminal cyclic nucleotide (cNMP) binding domain. The GNAT domain exhibits highest sequence identity to a protein N-acetyltransferase identified in S. enterica (15), while the N-terminal cNMP binding domain is most similar to the cNMP domain of eukaryotic protein kinases. The orthologue of Rv0998 in Mycobacterium smegmatis, MSMEG_5458, which shares 56% identity and 70% similarity based on the protein sequence analysis (Figure S1), has recently been shown to catalyze the acetylation of the universal stress protein (USP) using a glutathione-S-transferase pull-down assay (13). However, the lack of USP orthologues in other mycobacteria, including M. tuberculosis, suggests that Rv0998 and MSMEG_5458 have an alternative substrate that is unknown, and that acetylation of this substrate may be cNMP-dependent. Cyclic nucleotides are universal “second messengers” in both pro- and eukaryotic organisms, and cAMP was identified in extracts of several mycobacterial species in 1976 (16). While most prokaryotes contain a single adenylate cyclase (AC, e.g. E. coli) and some contain none (e.g. Bacillus species), mycobacteria, and in particular M. tuberculosis, contain over a dozen genes identified as AC’s, with many of these biochemically characterized in M. tuberculosis (reviewed in (17)). In M. tuberculosis, several of the AC’s have been shown to be specifically responsive to nitrogen and carbon limitation, pH and bicarbonate levels (18–21). Upon phagocytosis by macrophages, cAMP levels in M. tuberculosis have been shown to increase dramatically (22, 23). Restoration of basal cAMP levels is the result of hydrolysis of cAMP to 5′-AMP by cAMP phosphodiesterases, and secretion. In M. tuberculosis, the single annotated cAMP phosphodiesterase is only weakly active towards 3′,5′-cAMP and much more active with 2′,3′-cAMP (24), suggesting efflux as a major mechanism. Reports that cAMP efflux by M. tuberculosis into macrophages may produce immunosuppressive effects and reduction in cytokine production by infected macrophages have appeared (22). In this report, we attempted to identify a physiologically relevant substrate for MSMEG_5458 that would link production of cAMP to the acetylation of a protein substrate whose activity could be implicated in carbohydrate and amino acid metabolism. In addition, we sought the corresponding deacetylase (sirtuin), since it is universally recognized that post-translational protein modification coupled to activity activation/inactivation is a reversible phenomenon.