title: Mycobacterium tuberculosis Rv3586 (DacA) is a diadenylate cyclase that converts ATP or ADP into c-di-AMP.
abstract: 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.
authors.affiliation: Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.
authors.forename: Yinlan
authors.initials: Y
authors.lastname: Bai
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Xin
authors.initials: X
authors.lastname: Zhou
authors.forename: Xinxin
authors.initials: X
authors.lastname: Ding
authors.forename: Leslie E
authors.initials: LE
authors.lastname: Eisele
authors.forename: Guangchun
authors.initials: G
authors.lastname: Bai
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D015226:Dinucleoside Phosphates; D007477:Ions; D011994:Recombinant Proteins; C528998:cyclic diadenosine phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D019761:Phosphorus-Oxygen Lyases