C-di-GMP Hydrolysis by Pseudomonas aeruginosa HD-GYP Phosphodiesterases: Analysis of the Reaction Mechanism and Novel Roles for pGpG

Abstract

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.

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

“…Purified PA4781 did not contain iron in the active site as for previously reported HD-GYPs (14,16,18), and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 are in agreement with our previous data, indicating that this is preferentially a pGpG binding protein (15).…”

“…Nevertheless, a chaperone-mediated binding of a specific metal to PA4781 in vivo cannot be excluded (35). In our previous work, the enzymatic activity of recombinant PA4781 ("as purified") was measured only in the presence of Mn 2ϩ and Mg 2ϩ and was shown to be very low (15). Therefore, we measured the catalytic activity of PA4781 after reconstitution of the fully apo protein with Ni 2ϩ , Zn 2ϩ , Co 2ϩ , or the sole Mn 2ϩ in order to assess whether binding of other metals could significantly enhance the catalysis.…”

“…Indeed, it was shown that mutation of the GYP signature renders the protein RpfG unable to interact with its specific GGDEF partners (12), while leaving the catalytic activity unaffected (14). Moreover, it should be recalled that it is not excluded that degenerated HDGYPs (i.e., mutated in the HD-GYP signature), though catalytically inactive, may still function as c-di-GMP or even pGpG receptors (15,16). Recently, these puzzling issues benefited from the first two solved structures for this class of proteins.…”

“…Therefore, the authors of the latter structure suggested that the HD-GYP superfamily should be further divided in two subgroups, based on the presence of a bimetallic or a trimetallic center, and highlighted the need for further structural data to fully elucidate the features of this intriguing class of enzyme (14). We have recently presented a detailed biochemical characterization of two of the three HD-GYP proteins encoded by the genome of the human pathogen Pseudomonas aeruginosa (15), namely, PA4781 and PA4108, which were shown to affect c-di-GMP levels in vivo (the third, PA2572, displays a degenerated YN-GYP signature) (17). Here we move a step forward and report the crystal structure of PA4781, coupling a structural study to its previous in vitro characterization (15).…”

“…We have recently presented a detailed biochemical characterization of two of the three HD-GYP proteins encoded by the genome of the human pathogen Pseudomonas aeruginosa (15), namely, PA4781 and PA4108, which were shown to affect c-di-GMP levels in vivo (the third, PA2572, displays a degenerated YN-GYP signature) (17). Here we move a step forward and report the crystal structure of PA4781, coupling a structural study to its previous in vitro characterization (15). Strikingly, the structure shows a bimetallic active site whose metal binding mode is different from those of both Bd1817 and PmGH.…”