Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America.
PLoS One. 2012;7(4):e35206. doi: 10.1371/journal.pone.0035206. Epub 2012 Apr 17.
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.
环二鸟苷酸（c-di-GMP）和环二腺苷酸（c-di-AMP）是最近发现的信号分子。c-di-GMP 已被证明在细菌发病机制中发挥重要作用，而关于 c-di-AMP 的信息仍然非常有限。结核分枝杆菌 Rv3586（DacA）是枯草芽孢杆菌 DisA 的同源物，是一种推定的二腺苷酸环化酶。在本研究中，我们使用高效液相色谱法（HPLC）、质谱法（MS）和薄层层析法（TLC）在体外测定了 DacA 的酶活性。我们的结果表明，DacA 主要是一种二腺苷酸环化酶，类似于 DisA。此外，DacA 在体外还表现出残留的 ATP 酶和 ADP 酶活性。在所测试的潜在底物中，DacA 能够利用 ATP 和 ADP，但不能利用 AMP、pApA、c-di-AMP 或 GTP。通过使用凝胶过滤和分析超速离心，我们进一步证明 DacA 作为八聚体存在，其 N 端结构域有助于四聚化，C 端结构域提供额外的二聚化。N 端和 C 端结构域对于 DacA 的酶活性构象都是必不可少的。DacA 的二腺苷酸环化酶活性依赖于二价金属离子，如 Mg(2+)、Mn(2+)或 Co(2+)。DacA 在碱性 pH 下比在酸性 pH 下更活跃。DacA 中的保守 RHR 基序对于与 ATP 相互作用至关重要，该基序的突变 AAA 完全消除了 DacA 的二腺苷酸环化酶活性。这些结果为指定 DacA 为二腺苷酸环化酶提供了分子基础。我们未来的研究将探索该酶在结核分枝杆菌中的生物学功能。
