C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG.
In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.
Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy ; Department of Biochemical Sciences, Sapienza University of Rome, Rome, Italy.
Valentina
V
Stelitano
Giorgio
G
Giardina
Alessandro
A
Paiardini
Nicoletta
N
Castiglione
Francesca
F
Cutruzzolà
Serena
S
Rinaldo
D001426:Bacterial Proteins / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000378:metabolism
D001426:Bacterial Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP