Article Figures & Data
Figures
- 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; kc, rate constant for the chemical step.
- FIG. 2.
Loop 6 of RocR and the effects of mutations on catalysis. (A) Structural model of EALRocR 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 k 1, k 2, and k 3. (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.
Tables
- TABLE 1.
Steady-state kinetic parameters for RocR and its mutantsa
RocR enzyme kcat (s−1) K m (μM) kcat/K m (s−1 μM−1) K i (μM) Wild-type 0.67 ± 0.03 3.2 ± 0.3 0.21 ± 0.02 E268A mutant NDb ND ND E268Q mutant (1.5 ± 0.1) × 10−3 0.3 ± 0.1 (4.8 ± 0.5) × 10−3 S302A mutant 0.14 ± 0.07 5.5 ± 2.6 (2.5 ± 2.0) × 10−2 10.9 ± 3.0 D296A mutant (2.1 ± 0.3) × 10−2 8.6 ± 2.8 (2.7 ± 0.9) × 10−3 D56N D296A double mutant (1.2 ± 0.3) × 10−2 9.4 ± 2.3 (2.1 ± 0.6) × 10−3 D56N mutant 0.13 ± 0.01 0.2 ± 0.03 0.65 ± 0.1 F297A mutant 0.02 ± 0.01 0.7 ± 0.2 (2.8 ± 1.6) × 10−2
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
EAL domains with conserved catalytic residues and loop 6.
Zipped TXT file, 374K. - Supplemental file 2
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EAL domains with conserved catalytic residues and degenerate loop 6.
Zipped TXT file, 247K. - Supplemental file 3
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EAL domains with degenerate loop 6 and no catalytic residues.
Zipped TXT file, 120K. - Supplemental file 4
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EAL domains with conserved loop 6 and no catalytic residues.
Zipped TXT file, 10K.
- Supplemental file 1
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EAL domains with conserved catalytic residues and loop 6.
