Two-step synthesis and hydrolysis of cyclic di-AMP in Mycobacterium tuberculosis.
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.
Institute of Molecular Medicine, New Delhi, India.
Kasi
K
Manikandan
Council of Scientific and Industrial Research - Institute of Genomics and Integrative Biology, Delhi and IGIB Extension Centre (Naraina), New Delhi, India.
Varatharajan
V
Sabareesh
Central Instrument Facility, University of Delhi South Campus, New Delhi, India.
Nirpendra
N
Singh
Institute of Molecular Medicine, New Delhi, India.
Kashyap
K
Saigal
Institut Pasteur, CNRS UMR 3528, Unité de Biochimie des Interactions macromoléculaires, Paris, France.
Undine
U
Mechold
Institute of Molecular Medicine, New Delhi, India.
Krishna Murari
KM
Sinha
D000255:Adenosine Triphosphate / Q000737:chemistry; D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015226:Dinucleoside Phosphates / Q000096:biosynthesis*; D006868:Hydrolysis; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C528998:cyclic diadenosine phosphate; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D019761:Phosphorus-Oxygen Lyases