Two-Step Synthesis and Hydrolysis of Cyclic di-AMP in Mycobacterium tuberculosis

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.

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

“…They all hydrolyze c-di-AMP into linear dinucleotide 5Ј-pApA. There is also a PDE family that only contains a standalone DHH-DHHA1 domain, they hydrolyze c-di-AMP in a two-step process: first to linear 5Ј-pApA, which is subsequently hydrolyzed into two 5Ј-AMP molecules (16,17,20). All types of PDE enzymes require ions for catalytic activity.…”

An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

“…Listeria species encode a stand-alone DHH-DHHA1 protein, Lmo1575, homologous to other DHH-DHHA1 proteins that are reported to degrade c-di-AMP (9,12,13). Although PgpH is the only 7TMR_HD protein found in L. monocytogenes, seven other proteins of the HD superfamily (Pfam PF01966) are also present within the genome.…”

“…The first family comprises homologs of membrane-bound GdpP proteins, which contain a PAS domain, a degenerate GGDEF domain, and a catalytic DHH-DHHA1 domain (11). The second family comprises soluble, stand-alone DHH-DHHA1 domains (9,12,13).…”

Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes

“…The dried supernatants were solved with 100 or 200 l of H 2 O (depending on the expected amount of c-di-AMP). After repeated centrifugation and the addition of the internal standard 13 C, 15 N-c-di-AMP, part of the supernatants was analyzed by LC-MS/MS.…”

“…A recent study emphasized a function of DisA in controlling genome replication during outgrowth of B. subtilis spores to prevent propagation of damaged DNA to the germinating cells (12). Meanwhile, homologs of DisA have been identified in several other bacteria like Mycobacterium tuberculosis (13)(14)(15). It is interesting to note that some bacteria possess more than one DAC-containing protein (14,16).…”