The functional role of a conserved loop in EAL domain-based cyclic di-GMP-specific phosphodiesterase

Abstract

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (beta/alpha)(8) barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K(m)) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K(m) correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg(2+) ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

FIG. 1.

Kinetic model for mutants that exhibit substrate inhibition. The model assumes that the substrate can bind to the enzyme at a productive binding site and a nonproductive (inhibitory) binding site. Abbreviations: E, enzyme; P, product; S, substrate; k_c, rate constant for the chemical step.

FIG. 2.

Loop 6 of RocR and the effects of mutations on catalysis. (A) Structural model of EAL_RocR with the residues of loop 6 and Glu²⁶⁸ highlighted. The hydrogen bonds formed between Glu₂₆₈ and the loop residues Gly³⁰⁰ and Ser³⁰² are represented by the broken lines. The Mg²⁺ ion is shown as the ball. (B) Effects of the mutations in the loop 6 region on the steady-state kinetics of RocR. The curves were generated by fitting the data to the Michaelis-Menten equation with the exception of the S302A mutant, for which the curve was generated by fitting the kinetic data to equation 1.

FIG. 3.

Domain organization of the RocR protein, the PA2567 protein from P. aeruginosa PAO-1, and the DGC2 protein from A. xylinus (AxDGC2).

FIG. 4.

Effect of the E464A mutation on the steady-state kinetics of PA2567. The curves were generated by fitting the kinetic data of the wild-type and mutant enzyme to the Michaelis-Menten equation and equation 1, respectively.

FIG. 5.

Comparison of amide H/D exchange between RocR and the D56N mutant. (A) Sequence coverage map of RocR with the 21 peptides generated from pepsin digestion represented by the bars below the sequence. The peptides from the mutant exhibiting decrease in deuteration are colored blue, whereas the peptides showing significant and moderate increase in deuteration are colored red and yellow, respectively. (B) Structural models of the EAL and REC domains of RocR. The peptides exhibiting decrease in deuteration are colored blue, and the peptides showing significant and moderate increase in deuteration are colored red and yellow, respectively. The linker between the RR and EAL domains is represented by the brown broken line. (C) Time-dependent H/D exchange plots for the peptides in the EAL domain that exhibit changes in deuteration (RocR [•] and D56N mutant [○]). The curves were generated by fitting the data to equation 2 to obtain the total number of incorporated deuterons (N) as well as the exchange rate constants k₁, k₂, and k₃. (D) Time-dependent H/D exchange plots for the peptides in the phosphoreceiver (REC) domain and linker region that exhibit changes in deuteration (RocR ([•] and D56N mutant [○]). The phosphorylated site (Asp⁵⁶) and two putative residues (Ser⁸³ and Phe¹⁰⁵) involved in signal transduction are shown as sticks in panel B.

FIG. 6.

Enzymatic activity assay for the EAL domain of A. xylinus DGC2. (A) HPLC analysis of the enzymatic activity of the full-length A. xylinus DGC2, stand-alone EAL domain, and the triple mutant with three mutations (Gln⁴⁷³Lys⁴⁷⁶Ile⁴⁷⁸ → Asp⁴⁷³Thr⁴⁷⁶Tyr⁴⁷⁸) in the EAL domain. The enzymes were incubated with c-di-GMP and Mg²⁺ for 90 min. Abs (mAU), absorbance (milliabsorbance units). (B) Steady-state kinetics of the triple mutant of the stand-alone EAL domain. The curve was generated by fitting the data to the Michaelis-Menten equation.

FIG. 7.

Summary of EAL domains. (A) Comparison of loop 6 in active and inactive EAL domains. Loop 6 of TdEAL (Protein Data Bank accession no. 2r6o) and YkuI (Protein Data Bank accession no. 2w27) were highlighted in orange and cyan, respectively. The Mg²⁺ ion in the TdEAL structure is shown as a green ball, and c-di-GMP is shown as a stick. The corresponding residues of Asp²⁹⁵, Asp²⁹⁶, and Glu²⁶⁸ of RocR are highlighted and labeled (Asp⁶⁴⁶, Asp ⁶⁴⁷, and Glu⁶¹⁹ for TdEAL; Asp¹⁵², Asn¹⁵³, and Glu¹²⁵ for YkuI). (B) Comparison of the sequences of the loop among characterized EAL domains. The EAL domains shown are from the following proteins: PA2567, BifA, and FimX from Pseudomonas aeruginosa (indicated by the Pa suffix after the hyphen and protein) (16, 17, 31); TdEAL from Thiobacillus denitrificans (TdEAL-Td); VieA from Vibrio cholerae (VieA-Vc) (38); CC3396 from Crescentus caulobacter (CC3396-Cc) (9); YcgF, YahA, Dos, YciR, CsrD, and YegE from Escherichia coli (indicated by the Ec suffix after the hyphen and protein) (11, 32, 36, 40, 41); BphG from Rhodobacter sphaeroides (BphG-Rs) (39); PdeA1, DGC1, DGC2, and DGC3 from Gluconacetobacter xylinus (indicated by the Gx suffix after the hyphen and protein) (7, 37); HmsP from Yersinia pestis (Hmsp-Yp) (2); GcpC from Salmonella enterica (GcpC-Se) (10); STM1344 and STM3375 from Salmonella enterica serotype Typhimurium (indicated by the St suffix after the hyphen and protein)(33); and LapD from Pseudomonas fluorescens Pf0-1 (LapD-Pf) (24). The sequences were aligned using MultAlin (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html), and the figure was generated using ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/). (C) Pie chart summary of the classification of the 5,862 EAL domains from bacterial genomes according to the conservation of catalytic residues and loop 6.