Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications
Abstract
:1. Introduction
2. Results
2.1. Sequence and Structure Analysis
2.2. Overexpression and Purification of Recombinant PaIDH1, PaIDH2 and PaIDH K/P
2.3. Effects of pH, Temperature and Various Compounds on the Activity of PaIDH1 and PaIDH2
2.4. Kinetics Analysis
2.5. Evaluation of Coenzyme Specificity Determinants
2.6. In Vitro Phosphorylation and Identification of Phosphorylation Site
3. Discussion
4. Materials and Methods
4.1. Strains and Reagents
4.2. Plasmid Construction
4.3. Protein Expression and Purification
4.4. Enzyme Assay and Kinetic Studies
4.5. Effects of pH, Temperature and Compounds
4.6. Gel Filtration Chromatography
4.7. In Vitro Phosphorylation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Metabolite | Relative Activity (%) | |
---|---|---|
PaIDH1 | PaIDH2 | |
Control (none) | 100 ± 0.00 | 100 ± 0.00 |
ATP | 89.16 ± 5.17 | 75.91 ± 5.92 |
ADP | 81.79 ± 3.55 | 82.66 ± 3.37 |
AMP | 87.30 ± 1.95 | 91.17 ± 6.95 |
ATP + ADP | 79.04 ± 7.50 | 52.48 ± 4.76 |
AMP + ADP + AMP | 68.07 ± 6.59 | 59.61 ± 5.56 |
ATP + AMP | 83.89 ± 3.81 | 51.02 ± 5.04 |
ADP + AMP | 81.15 ± 5.73 | 80.68 ± 7.31 |
GTP | 85.13 ± 1.48 | 93.99 ± 1.3 |
GDP | 86.69 ± 1.16 | 88.43 ± 2.80 |
GMP | 83.32 ± 2.58 | 93.26 ± 3.09 |
GTP + GDP | 85.97 ± 1.38 | 90.88 ± 2.47 |
GTP + GMP | 85.74 ± 2.14 | 89.83 ± 4.12 |
GDP + GMP | 86.79 ± 1.66 | 87.63 ± 3.59 |
GTP + GDP + GMP | 84.43 ± 1.59 | 79.25 ± 4.96 |
CIA | 86.97 ± 4.95 | 94.81 ± 4.82 |
GLA | 93.05 ± 3.88 | 87.06 ± 3.73 |
α-KG | 79.52 ± 4.64 | 82.79 ± 5.65 |
OAA | 77.45 ± 4.56 | 88.32 ± 7.97 |
α-KG + OAA | 71.54 ± 4.76 | 70.7 ± 2.97 |
GLA + OAA | 75.71 ± 6.02 | 79.88 ± 7.08 |
α-KG + OAA + GLA | 68.17 ± 4.42 | 16.23 ± 0.56 |
α-KG + OAA + GLA + CIA | 68.53 ± 0.86 | 9.926 ± 1.22 |
Enzymes | ICT | NADP+ | References | ||||
---|---|---|---|---|---|---|---|
Km (μM) | kcat (s−1) | kcat/Km (μM−1s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1s−1) | ||
PaIDH1 | 14.41 ± 1.82 | 29.26 ± 10.92 | 2.10 ± 0.93 | 20.00 ± 6.29 | 104.51 ± 19.91 | 5.42 ± 0.97 | This study |
PaIDH2 | 23.61 ± 1.21 | 171.34 ± 6.77 | 7.26 ± 0.13 | 30.68 ± 2.73 | 133.98 ± 17.04 | 4.42 ± 1.13 | This study |
EcIDH | 24.2 ± 6.6 | 51.8 ± 4.4 | 2.1 | 17 | 80.5 | 4.7 | [8] |
MtIDH1 | 10 ± 5 | 3.8 | 0.38 | 125 ± 5 | 4 | 0.032 | [34] |
MtIDH2 | 20 ± 1 | 37.13 | 1.857 | 19.6 ± 6 | 37.4 | 1.908 | [34] |
AbIDH1 | 50.5 ± 3.2 | 50.4 | 1.0 | 46.6 ± 6.1 | 60.1 ± 3.3 | 1.3 | [37] |
AbIDH2 | 21 ± 3 | 39.2 ± 2.1 | 1.9 | 94 ± 6 | 36.9 ± 1.2 | 0.39 | [37] |
Enzymes | NADP+ | NAD+ | ||||
---|---|---|---|---|---|---|
Km (μM) | kcat (s−1) | kcat/Km (A) (μM−1s−1) | Km (μM) | kcat (s−1) | kcat/Km (B) (μM−1s−1) | |
PaIDH1 PaIDH1-D346 PaIDH1-D346I347 PaIDH1-D346I347A353 PaIDH1-D346I347A353K393 PaIDH1-K393 | 20.00 ± 6.29 1715.67 ± 133.5 - - - 74.36 ± 5.28 | 104.51 ± 19.91 39.56 ± 2.43 - - - 41.32 ± 1.95 | 5.42 ± 0.97 0.02 ± 0.003 - - - 0.56 ± 0.04 | - 15,368.79 ± 2818 - 4500 ± 157.36 925.1 ± 83.81 - | - 0.003 ± 0.0003 - 4.73 ± 0.76 7.04 ± 0.71 - | - 0.02 × 10−6 - 0.001 ± 0.0002 0.008 ± 0.001 - |
Enzymes | NADP+ | NAD+ | ||||
---|---|---|---|---|---|---|
Km (μM) | kcat (s−1) | kcat/Km (A) (μM−1s−1) | Km (μM) | kcat (s−1) | kcat/Km (B) (μM−1s−1) | |
PaIDH2 | 30.68 ± 2.73 | 13 3.98 ± 17.04 | 4.42 ± 1.13 | - | - | - |
PaIDH2-L589 | 135.33 ± 14.4 | 52.36 ± 9.08 | 0.39 ± 0.1 | 5927.3 ± 205.25 | 10.89 ± 0.15 | 0.002 ± 0.00008 |
PaIDH2-L589 D600 | 849.33 ± 71.92 | 1.7 ± 0.35 | 0.002 ± 0.0004 | 8949.33 ± 748.06 | 4.5 ± 0.41 | 0.0005 ± 0.000003 |
PaIDH2-L589 D600 S649 | - | - | - | 12242.33 ± 497.22 | 6.63 ± 0.12 | 0.0005 ± 0.00001 |
PaIDH2-L589 I600 | 7770.67 ± 270.55 | 16.03 ± 0.79 | 0.002 ± 0.00005 | 5824.33 ± 346.01 | 20.74 ± 0.43 | 0.004 ± 0.0002 |
PaIDH2-L589 I600 L649 | - | - | - | 15625.33 ± 1751 | 28.92 ± 2.73 | 0.002 ± 0.0001 |
PaIDH2-L589 I600 D649 | - | - | - | 9968.67 ± 1051.5 | 48.19 ± 4.15 | 0.005 ± 0.0002 |
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Chen, X.; Wei, W.; Xiong, W.; Wu, S.; Wu, Q.; Wang, P.; Zhu, G. Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications. Int. J. Mol. Sci. 2023, 24, 14985. https://doi.org/10.3390/ijms241914985
Chen X, Wei W, Xiong W, Wu S, Wu Q, Wang P, Zhu G. Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications. International Journal of Molecular Sciences. 2023; 24(19):14985. https://doi.org/10.3390/ijms241914985
Chicago/Turabian StyleChen, Xuefei, Wei Wei, Wei Xiong, Shen Wu, Quanchao Wu, Peng Wang, and Guoping Zhu. 2023. "Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications" International Journal of Molecular Sciences 24, no. 19: 14985. https://doi.org/10.3390/ijms241914985
APA StyleChen, X., Wei, W., Xiong, W., Wu, S., Wu, Q., Wang, P., & Zhu, G. (2023). Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications. International Journal of Molecular Sciences, 24(19), 14985. https://doi.org/10.3390/ijms241914985