A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP
Abstract
1. Introduction
2. Materials and Methods
2.1. Plasmids and Strains
2.2. Recombinant Enzyme Expression
2.3. Enzyme Purification
2.4. Enzyme Cascade Reactions
2.5. In Vitro Enzyme Assays
2.6. Quantification of 2′3′-cGAMP, Adenosine, AMP, ADP, ATP, and GTP
3. Results and Discussion
3.1. 2′3′-cGAMP Synthesis by a Multi-Enzyme Cascade Reaction
3.2. Specific Activities of the Kinases
3.3. Adaptation of the Assay Composition in the Multi-Enzyme Cascade Reaction
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Enzyme | Enzyme Concentration | Reaction Rate (μmol L−1 min−1) | Specific Activity (U mg−1) | |
---|---|---|---|---|
(mg L−1) | (nM) | |||
ScADK | 5 | 130 | 21.7 ± 4.4 | 4.3 ± 0.9 |
AjPPK2 | 0.5 | 9 | 34.6 ± 5.4 | 69.2 ± 10.8 |
SmPPK2 | 5 | 140 | 13.8 ± 3.1 | 2.8 ± 0.6 |
ScADK:AjPPK2: SmPPK2:thscGAS Ratio | 2′3′-cGAMP Reaction Rates (μmol L−1 min−1) | thscGAS Specific Activity (U mg−1) | (mol2′3′-cGAMP molAdo−1) | Enzyme Usage (mg2′3′-cGAMP mgenzyme−1) | |
---|---|---|---|---|---|
(mg:mg:mg:mg) | (µmol:µmol:µmol:µmol) | ||||
50:50:50:40 | 1.3:0.9:1.4:0.7 | 2.10 ± 0.60 | 0.052 ± 0.014 | 0.030 ± 0.001 | 1.12 ± 0.04 |
50:5:50:120 | 1.3:0.09:1.4:2.1 | 1.42 ± 0.50 | 0.012 ± 0.004 | 0.070 ± 0.004 | 1.87 ± 0.19 |
5:0.5:5:120 | 0.13:0.009:0.14:2.1 | 0.25 ± 0.14 | 0.002 ± 0.001 | 0.077 ± 0.030 | 0.75 ± 0.03 |
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Becker, M.; Nikel, P.; Andexer, J.N.; Lütz, S.; Rosenthal, K. A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP. Biomolecules 2021, 11, 590. https://doi.org/10.3390/biom11040590
Becker M, Nikel P, Andexer JN, Lütz S, Rosenthal K. A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP. Biomolecules. 2021; 11(4):590. https://doi.org/10.3390/biom11040590
Chicago/Turabian StyleBecker, Martin, Patrick Nikel, Jennifer N. Andexer, Stephan Lütz, and Katrin Rosenthal. 2021. "A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP" Biomolecules 11, no. 4: 590. https://doi.org/10.3390/biom11040590
APA StyleBecker, M., Nikel, P., Andexer, J. N., Lütz, S., & Rosenthal, K. (2021). A Multi-Enzyme Cascade Reaction for the Production of 2′3′-cGAMP. Biomolecules, 11(4), 590. https://doi.org/10.3390/biom11040590