Catalytic Mechanism of Cyclic Di-GMP-Specific Phosphodiesterase: a Study of the EAL Domain-Containing RocR fromPseudomonas 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.

FimK regulation on the expression of type 1 fimbriae in Klebsiella pneumoniae CG43S3

“…S1, the FimK EIL domain includes the critical residues involved in Mg 2+ substrate binding and Loop 6 required for signal transduction (Rao et al, 2008(Rao et al, , 2009Römling, 2009). This suggests PDE activity for FimK.…”

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…We further defined the c-di-GMP binding site of LapD by constructing alanine replacements in candidate residues chosen for their involvement in substrate binding and their proximity to the active site of the well-characterized PDE RocR (22). LapD variants with the mutations K446A, R450A, K581A, and E617A were stably expressed in the membrane in vivo at WT levels ( Fig.…”

Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes

“…atrosepticum genome sequence (accession no. BX950851) that contain the GGDEF, EAL and HD-GYP domains, respectively (Aldridge et al, 2003;Rao et al, 2008;Ryan et al, 2006). These searches identified 23 putative c-di-GMP metabolism proteins encoded by the genome of Pe.…”

“…Active EAL domains are characterized by the presence of seven key residues and a consensus loop 6, found to be important for stabilization of the catalytic domain (Rao et al, 2008(Rao et al, , 2009). These active EAL domains require Mg 2+ or Mn 2+ to catalyse the degradation of c-di-GMP to a linear intermediate, 59-phosphoguanylyl-(39-59)-guanosine (pGpG).…”