The Cyclic Dinucleotide c-di-AMP Is an Allosteric Regulator of Metabolic Enzyme Function

Abstract

SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide.

An Essential Poison: Synthesis and Degradation of Cyclic Di-AMP in Bacillus subtilis

“…However, the function of this protein has so far remained elusive (17,18,24). Finally, c-di-AMP controls the activity of the pyruvate carboxylase in L. monocytogenes (18). However, all these identified targets cannot answer the question of why c-di-AMP is essential in the firmicutes.…”

“…In all firmicutes that have been studied so far, c-di-AMP binds to DarA, a small PII-like regulatory protein. However, the function of this protein has so far remained elusive (17,18,24). Finally, c-di-AMP controls the activity of the pyruvate carboxylase in L. monocytogenes (18).…”

“…CdaA is the most widespread diadenylate cyclase, and it is the only such enzyme in pathogenic firmicutes. CdaA has been implicated in the control of cell wall and potassium homeostasis (6,(16)(17)(18)(19). The cdaA gene is the first gene of a widely conserved gene cluster that also encodes a regulatory protein, CdaR, and enzymes that generate glucosamine-1- …”

Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation

“…Comparison of the positions of these residues in the RePC tetramer structure determined with acetyl CoA bound with those in the structure of RePC tetramer without acetyl CoA bound is not possible because of the low resolution of the latter structure [6,7]. The crystal structures of other pyruvate carboxylase tetramers that have been determined have not exhibited this very marked asymmetry [8][9][10]16]. The crystal structure of SaPC has been determined in the absence of acetyl CoA (PDB: 3BG5) and also in its presence (PDB: 3H08), where the activator is bound to all four subunits.…”

“…In addition, the cooperativity of the activation of PC by acetyl CoA varies between species [3], with the Hill coefficient for the activation of the Rhizobium etli enzyme (RePC) being about 2.8 [5]. In recent years, the structures of the a 4 tetrameric pyruvate carboxylase holoenzyme from R. etli, Staphylococcus aureus (SaPC) and Listeria monocytogenes (LmPC) have been solved [6][7][8][9][10] and the catalytic mechanism of the RePC has been well characterised [11][12][13][14][15]. However, many aspects of the mechanism of action of acetyl CoA in its role as an allosteric activator of RePC remain to be established.…”