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 5'pGpG. 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 5'pGpG 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.

FIG. 1.

Overexpression and purification of the E. coli YahA protein, the EAL domain of YahA, and the EAL domain of E. coli Dos (protein chip, Bioanalyzer). Lane 1, molecular mass markers (molecular masses [in kDa] are indicated on the left); lanes 2, 5, and 8, crude extracts of E. coli DH5α cells prior to induction of expression of the fusion proteins; lanes 3, 6, and 9, crude extracts of E. coli cells after 2 h of induction with IPTG; lanes 4, 7, and 10, purified recombinant proteins.

FIG. 2.

HPLC chromatograms showing the progression (0, 10 s, 2 min, 60 min) of c-di-GMP hydrolysis by YahA (final concentration of protein, 5 μM). For quantification purposes, the same amount of NAD was added to each sample prior to injection into the column.

FIG. 3.

(A) pH dependence of the PDE-A activity of YahA. (B) Dependence of the PDE-A activity of YahA on divalent metal cations. The PDE-A reaction buffer (pH 9.35) contained different divalent metal cations (final concentration, 5 mM) instead of Mg²⁺. The bar on the right shows inhibition of the PDE-A activity in the standard reaction buffer containing 5 mM (final concentration) MgCl₂ or 5 mM (final concentration) MnCl₂ by Ca²⁺ (5 mM [final concentration] CaCl₂). The reaction with Co²⁺ was carried out in PDE buffer at pH 8.4 to avoid precipitation of Co²⁺ complexes at higher pH values. The error bars indicate the standard deviations calculated from at least three replicates.

FIG. 4.

Lineweaver-Burk double-reciprocal plots for YahA (A), the EAL domain of YahA (B), and the EAL domain of Dos (C), showing the dependence of the initial reaction rate (V) on the substrate concentration (S). The initial rates of the reactions were calculated using the dependence of l-di-GMP formation on time. The plot showing this dependence for EAL_YahA is present in panel B for illustrative purposes; the initial c-di-GMP concentration (μM) for each curve is indicated on the right.

FIG. 5.

Biochemical analysis of l-di-GMP. G, guanosine; AP, shrimp alkaline phosphatase; MBN, mung bean nuclease.

FIG. 6.

Two proposed classes of EAL domains, enzymatically active and inactive. The multiple-sequence alignment of the enzymatically active EAL domains (above the consensus line) was generated by using Clustal W (11) and was adjusted manually. Residues identical in >80% of the active EAL domains are indicated by a black background. Residues conserved in 100% of the active EAL domains are indicated in the consensus line by asterisks. Similar residues present in 100% of the active EAL domains are indicated by a grey background. The proposed enzymatically inactive EAL domains are below the consensus line. Proteins whose activities have been tested in vitro are indicated by boldface type. Ec, E. coli; Gx, G. xylinus; Rs, R. sphaeroides; Se, S. enterica; Vc, V. cholerae.

FIG. 7.

Substrate specificity of the EAL domain of Dos (final concentration, 0.2 μM). (A) c-di-GMP; (B) cAMP.