4 research outputs found
Mycobacterium tuberculosis Rv3586 (DacA) Is a Diadenylate Cyclase That Converts ATP or ADP into c-di-AMP
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 Mg2+, Mn2+ or Co2+. 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
SERS and NMR Studies of Typical Aggregation-Induced Emission Molecules
Over recent decades, aggregation-induced
emission (AIE) molecules
have attracted increasing attention. Restriction of intramolecular
rotation (RIR) has been widely accepted as the cause of the emission
when AIE molecules aggregate into clusters. The intramolecular rotation
of AIE molecules can be monitored by molecular vibration spectra such
as nuclear magnetic resonance (NMR), infrared, and Raman, especially
surface-enhanced Raman scattering (SERS) which has high sensitivity
down to a single molecule. We employed SERS and NMR to study the AIE
emission mechanism and compared experimental results with simulation
data to monitor the RIR. Interestingly, we found that intramolecular
rotation was also restricted for individual AIE molecules loaded onto
SERS substrate surfaces due to the laid-down configuration