Article Figures & Data
Figures
- FIG. 1.
Phosphodiesterase A (PDE-A) catalyzes hydrolysis of cyclic-di-GMP to generate the linear diguanylic acid 5′-pGpG.
- FIG. 2.
Structural model of the EALRocR domain with the 14 conserved polar residues highlighted. The four residues for Mg2+ ion (sphere) binding are shown in red. The two distal residues are in green, whereas the rest of the conserved residues are in yellow.
- FIG. 3.
Recovery of catalytic activity at elevated Mg2+ concentrations. The assay conditions were as follows: 100 mM Tris buffer (pH 8.0), 20 mM KCl, ∼1.0 μM enzyme, 20 μM cyclic di-GMP, and 20-min incubation. WT, wild type.
- FIG. 4.
pH dependence of enzymatic activity for wild-type (WT) RocR and the mutant E352Q. The curves were obtained from the nonlinear-least-square fitting of the data to equation 1 (see Materials and Methods).
- FIG. 5.
Model of the tdEAL-Mg2+-substrate ternary complex generated from computational docking. The residue numbers for tdEAL are shown, along with the corresponding residue numbers for EALRocR in parentheses. The water (W) (yellow ball) is coordinated by Mg2+ (green ball) and hydrogen bonded to E182 (D295) and E239 (E352) for deprotonation and nucleophilic attack. The water is positioned 3.0 Å away from the electrophilic phosphorus center, with the leaving group (OL) aligned linearly on the opposite side of the phosphorus.
- FIG. 6.
Interaction between E268 and the loop between β5 and α6. Residues from the loop (F297 GAG300 YS302), E268, and the two neighboring residues (D295 and E265) for Mg2+ ion (green ball) binding are highlighted to show that mutation of E268 may affect Mg2+ and substrate binding.
- FIG. 7.
Sequence alignment of the EAL domains with characterized catalytic activity. The numbering of the residues is based on the RocR sequence, and the secondary structure is based on the structure of tdEAL (PDB ID, 2R6O). EALYkuI and tdEAL are included as active EAL domains. The inactive EAL domains are shown in the boxes. The residues examined in this study are indicated by black and red asterisks (essential residues). The loop that interacts with E268 is underlined. Shown are PA2567, BifA, and FimX from P. aeruginosa (16, 19, 30); TdEAL from T. denitrificans; VieA from V. cholerae (38); CC3396 from C. crescentus (6); YahA, Dos, YciR, CsrD, and YegE from E. coli (11, 31, 35, 41); BphG from Rhodobacter sphaeroides (39); PdeA1, DGC1, DGC2, and DGC3 from G. xylinus (5, 36); HmsP from Yersinia pestis (2); GcpC from S. enterica (9); and STM1344 and STM3375 from S. enterica serovar Typhimurium (32). 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/ ).
Tables
- TABLE 1.
Steady-state kinetic parameters for RocR and its mutantsa
Enzyme kcat (s−1) Km (μM) kcat/Km (s−1 μM−1) Decrease in kcat (fold) RocR 0.67 ± 0.03 3.2 ± 0.3 0.21 Q161A 0.13 ± 0.01 6.3 ± 1.0 (2.1 ± 0.4) × 10−2 5.1 E175A NDb >105 R179A (2.3 ± 0.1) × 10−2 8.0 ± 0.9 (2.9 ± 0.3) × 10−3 29.1 N233A ND >105 E265A ND >105 T267A (4.9 ± 0.1) × 10−2 2.2 ± 0.4 (2.2 ± 0.4) × 10−2 13.4 E268A ND >105 E268Q (1.5 ± 0.1) × 10−3 0.3 ± 0.1 (4.8 ± 0.5) × 10−3 446 D295A ND >105 D296A (2.1 ± 0.3) × 10−2 8.6 ± 2.8 (2.7 ± 0.9) × 10−3 33.5 K316A ND >105 D318A (8.0 ± 0.5) × 10−2 5.9 ± 1.5 (1.4 ± 0.4) × 10−2 8.4 E352A ND >105 E352C ND >105 E352Q (1.1 ± 0.1) × 10−5 3.8 ± 0.7 (2.9 ± 0.6) × 10−6 6.1 × 104 E352D (2.2 ± 0.1) × 10−5 2.2 ± 0.5 (1 ± 0.2) × 10−5 3.0 × 104 E355A 0.51 ± 0.07 2.6 ± 1.1 0.19 ± 0.09 1.3 Q372A (8.0 ± 0.5) × 10−2 1.3 ± 0.3 (6.2 ± 1.5) × 10−2 8.4
