Mycobacterium tuberculosis Rv3586 (DacA) is a diadenylate cyclase that converts ATP or ADP into c-di-AMP

Queen Mary University of London, United Kingdom
PLoS ONE (Impact Factor: 3.23). 04/2012; 7(4):e35206. DOI: 10.1371/journal.pone.0035206
Source: PubMed


Cyclic diguanosine monophosphate (c-di-GMP) and cyclic diadenosine monophosphate (c-di-AMP) are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA), which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC), mass spectrometry (MS) and thin layer chromatography (TLC). Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg(2+), Mn(2+) or Co(2+). DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.

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    • "The DAC activity of MtbDisA has been reported before [21] but we have not been able to get any activity under the conditions reported. We have rather found that 2 mM of Mn2+ and ATP are inhibitory for the activity. "
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    ABSTRACT: Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5'-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5'-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5'-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "c-di-AMP is synthesized by a protein DacA, which contains a di-adenylate cyclase domain (DAC domain) [5–8]. The production of c-di-AMP seems to be essential for viability because the DAC domain protein cannot be deleted using traditional genetic techniques in bacteria such as Staphaureus [5], Mycoplasma genitalium [9], Mycobacteriumpulmonis [10], Streptococcspneumoniae [11], and L. monocytogenes [7] that possess only one DAC protein. "
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    ABSTRACT: Cyclic dinucleotides, including cyclic-di-AMP, are known to be ubiquitous second messengers involved in bacterial signal transduction. However, no transcriptional regulator has been characterized as a c-di-AMP receptor/effector to date. In the present study, using a c-di-AMP/transcription factor binding screen, we identified Ms5346, a TetR family regulator in M. smegmatis, as a c-di-AMP receptor in bacteria. Ms5346 could specifically bind c-di-AMP with Kd of 2.3 μM. Using EMSA and DNase I footprinting assays, c-di-AMP was found to stimulate the DNA-binding activity of Ms5346 and to enhance its ability to protect its target DNA sequence. A conserved 14-bp palindromic motif was identified as the DNA binding site for Ms5346. Further, Ms5346 was found to negatively regulate expression of three target genes including Ms5347 (encoding a major facilitator family transporter), Ms5348 (encoding a medium-chain fatty acyl-CoA ligase), and Ms5696 (encoding a cold shock protein, CspA). Ms5346 is the first cyclic-di-AMP receptor regulator to be identified in bacteria, and we have designated it as DarR. Our findings enhance our understanding of the function and regulatory mechanism of the second messenger c-di-AMP in bacteria.
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