Abstract
Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth. It has been found mainly in Gram-positive bacteria, including pathogenic bacteria like Listeria monocytogenes CdaA is the sole diadenylate cyclase in L. monocytogenes, making this enzyme an attractive target for the development of novel antibiotic compounds. Here we report crystal structures of CdaA from L. monocytogenes in the apo state, in the post-catalytic state with bound c-di-AMP and catalytic Co2+ ions, as well as in a complex with AMP. These structures reveal the flexibility of a tyrosine side chain involved in locking the adenine ring after ATP binding. The essential role of this tyrosine was confirmed by mutation to Ala, leading to drastic loss of enzymatic activity.
Highlights
Cyclic di-AMP (c-di-AMP) is the only second messenger known to be essential for bacterial growth
Cyclic di-AMP (c-di-AMP)2 is the most recently discovered bacterial signaling nucleotide and, to date, has been found mostly in Gram-positive bacteria. c-di-AMP is involved in different cellular processes, such as DNA integrity scanning, cell wall metabolism, and osmolyte homeostasis. c-di-AMP is the only essential second messenger in bacteria because of its role in potassium homeostasis
CdaA of the human pathogen L. monocytogenes appears to be an attractive target, as c-di-AMP synthesis is essential for bacterial growth and CdaA is the only diadenylate cyclase (DAC) in this pathogenic bacterium, whereas there are no DACs in humans
Summary
Structure-based development of novel antibiotic drugs requires high-resolution three-dimensional structures of the targeted enzyme and enzyme–inhibitor complexes. We have demonstrated previously that this truncated ⌬100CdaA has preserved its enzymatic activity with a higher enzymatic activity for Co2ϩ compared with Mn2ϩ but no activity for of Mg2ϩ [15] In this previous study, the in vitro activity was measured by LC-MS/MS, we applied a direct fluorescence-based measurement of c-di-AMP formation by its binding to coralyne [16]. Careful inspection of the difference electron density map revealed a small molecule bound to the surface of one of two CdaA molecules in the asymmetric unit (Fig. S1). This electron density was interpreted as a sucrose molecule originating from the utilized cryo-protectant solution. In the apo-CdaA crystal structure, the active site is accessible from solvent channels; this crystal form of apo-CdaA appears to be suitable for a fragment screen
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