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Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa.

ABSTRACT: EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg(2+) ion. In addition to the five essential residues involved in Mg(2+) binding, we propose that the essential residue E(352) functions as a general base catalyst assisting the deprotonation of Mg(2+)-coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k(cat) or K(m), leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.

SUBMITTER: Rao F

PROVIDER: S-EPMC2394985 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa.

Rao Feng F Yang Ye Y Qi Yaning Y Liang Zhao-Xun ZX

Journal of bacteriology 20080314 10

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm for ...[more]

PMID: 18344366

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Project description:Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250?000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.

Dataset Information

Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa.

Publications

Catalytic mechanism of cyclic di-GMP-specific phosphodiesterase: a study of the EAL domain-containing RocR from Pseudomonas aeruginosa.

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