%0 Journal Article %T C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG. %+ Department of Biochemical Sciences "Rossi Fanelli" %+ Department of Biochemical Sciences %A Stelitano, Valentina %A Giardina, Giorgio %A Paiardini, Alessandro %A Castiglione, Nicoletta %A Cutruzzolà, Francesca %A Rinaldo, Serena %Z This work was supported by Ministero della Universita e Ricerca of Italy [RBRN07BMCT and 20094BJ9R7 to FC, RBFR10LHD1 to SR];Sapienza University of Rome to FC and AP; Fondazione Italiana Fibrosi Cistica [13/2009- with the contribution of Delegazione Novara and Delegazione Cosenza2 to FC]. VS acknowledges a Post-doctoral fellowship on the project C26A10NXKK funded by Sapienza University of Rome. %< avec comité de lecture %@ 1932-6203 %J PLoS ONE %I Public Library of Science %V 8 %N 9 %P e74920 %8 2013 %D 2013 %R 10.1371/journal.pone.0074920 %M 24066157 %Z Life Sciences [q-bio]/Biochemistry, Molecular BiologyJournal articles %X 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. %G English %2 https://riip.hal.science/pasteur-01048756/document %2 https://riip.hal.science/pasteur-01048756/file/Stelitano_PlosOne.pdf %L pasteur-01048756 %U https://riip.hal.science/pasteur-01048756 %~ RIIP %~ RIIP_FCB