Catalytic Mechanism of C-di-GMP Specific Phosphodiesterase: A Study of the EAL Domain-Containing RocR from Pseudomonas Aeruginosa

Division of Chemical Biology & Biotechnology, Division of Computational and Structural Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551

^* To whom correspondence should be addressed. Email: zxliang{at}ntu.edu.sg.

	Abstract

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second messenger c-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 c-di-GMP signaling network. Herein, 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 c-di-GMP by using a general base-catalyzed mechanism with the assistance of an Mg²⁺ ion. In addition to the five essential residues involved in Mg²⁺ binding, we propose that the essential residue E₃₅₂ functions as a general base catalyst assisting the deprotonation of an Mg²⁺-coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in k_cat or K_m, leading us to propose their roles in residue positioning and substrate binding. With functions assigned for the conserved groups in the active site, we discuss the molecular basis for the lack of activity for some characterized EAL domain proteins, and the possibility of predicting the phosphodiesterase activity of the vast number of EAL domains in bacterial genomes in the light of the catalytic mechanism.