The Ubiquitous Protein Domain EAL Is a Cyclic Diguanylate-Specific Phosphodiesterase: Enzymatically Active and Inactive EAL Domains

Abstract

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on Mg 2؉ or Mn 2؉ , strongly inhibited by Ca 2؉ , and independent of protein oligomerization. Linear dimeric GMP was shown to be 5pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.

Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence

“…The levels of c-di-GMP are also controlled by c-di-GMP phosphodiesterases (PDEs) that contain a conserved EAL domain (4). A number of proteins contain both of these domains, suggesting they may be bifunctional enzymes with opposing activities, although the EAL domains of these presumptive bifunctional proteins tend to be more divergent and perhaps inactive (5).…”

The structure–function relationship of WspR, a Pseudomonas fluorescens response regulator with a GGDEF output domain

“…Although the biological pathways through which c-di-GMP regulates persistence, cell aggregation and the switch to the commensal lifestyle are currently poorly understood, the proteins responsible for c-di-GMP synthesis and degradation have been determined and, at least partially, characterized. Bacterial diguanylate cyclase (DGC) activity is found in GGDEF domain-containing proteins (Paul et al, 2004;Ryjenkov et al, 2005;Hickman et al, 2005;Kulesekara et al, 2006), whilst EAL domain-containing proteins have been shown to have c-di-GMP-specific phosphodiesterase (PDE) activity (Bobrov et al, 2005;Christen et al, 2005;Schmidt et al, 2005;Kulesekara et al, 2006).…”

Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella

“…The only known effector domain that synthesizes c-di-GMP (designated as diguanylate cyclase activity, DGC) is the GGDEF domain. Two domains, EAL and HD-GYP, hydrolyze c-di-GMP (designated as phosphodiesterase activity, PDE) into linear 5Ј-phosphoguanylyl-(3Ј-5Ј)-guanosine (pGpG) or guanosine monophosphate (GMP), respectively (7)(8)(9)(10)(11)(12)(13). Many DGC proteins also contain an RxxD conserved motif 5 amino acids upstream from the GGDEF motif.…”