Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications

Abstract

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP⁺-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP⁺ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD⁺ while it lost activity towards NADP⁺, and the Km value (7770.67 μM) of the mutant PaIDH2-L589 I600 for NADP⁺ was higher than that observed for NAD⁺ (5824.33 μM), indicating a shift in coenzyme specificity from NADP⁺ to NAD⁺ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

Keywords: Pseudomonas aeruginosa; coenzyme specificity; evolution; isocitrate dehydrogenase; phosphorylation.

Figure 1

Structure-based protein sequence alignment of PaIDH with other IDHs. Putative coenzyme binding sites are marked with triangles. Phosphorylation site of PaIDH1 is marked with circle. The completely conserved residues are shaded in red. The secondary structure elements are placed above the alignment. Image created by ESPript 3.0 [24]. (A) The deduced conserved residues involved in PaIDH1 coenzyme binding were aligned with prokaryotic NADP-IDHs (Escherichia coli IDH and B. subtilis IDH) and prokaryotic NAD-IDHs (Acidithiobacillus thiooxidans IDH and Zymomonas mobilis IDH) based on the PaIDH1 structure (PDB entry 5m2e) and AtIDH structure (PDB entry 2d4v). (B) The deduced conserved residues involved in PaIDH2 coenzyme binding were aligned with prokaryotic NADP-IDHs (Azotobacter vinelandii IDH, Corynebacterium glutamicum IDH and Acinetobacter baumannii IDH2) and prokaryotic NAD-IDHs (Campylobacter curvus IDH and Campylobacter pinnipediorum IDH) based on the PaIDH2 structure (PDB entry 6g3u). (C) Comparison of PaIDH1 phosphorylation sites with other prokaryotes based on the PaIDH1 structure (PDB entry 5m2e).

Figure 2

Phylogenetic analysis of IDHs from different species. The analysis involved 38 IDH sequences and a neighbor-joining tree with 1000 bootstraps and was created by MEGA 7.0. PaIDHs are marked by red stars.

Figure 3

Molecular modeling of PaIDH1 and PaIDH2. (A) Binding model of PaIDH1 with coenzyme NADP⁺. On the left, a superimposition illustrates the overall structure of modeled PaIDH1 in green, with the PaIDH1 model generated using the Swiss-model server and E. coli IDH in cyan (PDB code: 4aj3) as a template. The magnified view on the right highlights critical determinants of coenzyme specificity, depicted as thick sticks. Site-directed mutagenesis targeted Lys346, Tyr347, Val353 and Tyr393. (B) Binding model of PaIDH2 with coenzyme NADP⁺. On the left, a superimposition presents the overall structures of modeled PaIDH2 in green (PDB code: 6g3u). The enlarged view on the right highlights essential determinants of coenzyme specificity, represented as thick sticks. Site-directed mutagenesis focused on His589, Arg600 and Arg649.

Figure 4

Determination of the molecular mass of PaIDH1 (A), PaIDH2 (B,D) and PaIDH K/P (C). The flow rate was 0.5 mL/min and the proteins in the fractions were monitored at 280 nm. (A) Result of gel filtration chromatography of PaIDH1. (B) Result of gel filtration chromatography of PaIDH2. (C) Result of gel filtration chromatography of PaIDH K/P. (D) Result of gel filtration chromatography of PaIDH2 at different salt concentrations. The elution volume was observed to be 11.12 mL at a NaCl concentration of 0.1 M, 11.57 mL at a NaCl concentration of 1 M and 11.75 mL at a NaCl concentration of 3 M. The experiment was conducted three times in accordance with the principles of biology.

Figure 5

Effects of pH and temperature on the activity of purified PaIDH1 and PaIDH2. (A) Effects of pH range of 6.8–8.8 on PaIDH1 (■) and PaIDH2 (●), respectively. (B) Effects of temperature range of 25–60 °C on PaIDH1 (■) and PaIDH2 (●), respectively. (C) Heat inactivation profiles of PaIDH1 and PaIDH2. The PaIDH1 (■) and PaIDH2 (●) activity was measured from 25 to 55 °C, respectively.

Figure 6

Identification of the phosphorylated PaIDH1 and PaIDH2. (A) Identification of the phosphorylated PaIDH1 and PaIDH2 in vitro using Phos-tag SDS-PAGE. Lane 1, 6: PaIDH K/P as the control. Lane 2: PaIDH1 treated with PaIDH K/P. Lane 3: PaIDH1 untreated with PaIDH K/P as the control. Lane 4: PaIDH2 untreated with PaIDH K/P as the control. Lane 5: PaIDH2 treated with PaIDH K/P. (B) In vitro phosphorylation of PaIDH1 and PaIDH2 by EcIDH K/P and PaIDH K/P. (C) Mass spectrometry detection of PaIDH1 phosphorylation site. (S) indicates a phosphorylation site.