2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design of a Fe(II)/2-KG DO-Based In Vitro Enzymatic Biosystem for Hydroxy Amino Acid Synthesis
2.2. Enzyme Kinetics
2.3. Effects of Reaction Components on IDO and LGOX Activity
2.4. Enzymatic Cascade for 4-HIL Synthesis
2.5. Fed-Batch 4-HIL Synthesis Reaction
2.6. In Vitro Enzymatic Biosystem for Hydroxy Amino Acids Synthesis
3. Materials and Methods
3.1. Reagents
3.2. Preparation of Recombinant Enzymes
3.3. Enzymatic Activity Assays
3.4. Determination of Km and Vmax
3.5. Effects of Reaction Components on Enzyme Activities
3.6. One-Pot Synthesis of 4-HIL
3.7. Two-Step Process for Hydroxy Amino Acid Synthesis
3.8. Fed-Batch Reaction for 4-HIL Synthesis
3.9. Analysis of Organic Acids
3.10. Analysis of Amino Acids
3.11. Nuclear Magnetic Resonance (NMR) Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Enzyme | Substrate | Km (mM) | Vmax (U·mg−1) | kcat (s−1) | kcat/Km (s−1·m·M−1) |
---|---|---|---|---|---|
IDO | l-Ile | 6.34 ± 0.12 | 8.99 ± 0.09 | 4.18 ± 0.08 | 0.66 ± 0.04 |
2-KG | 15.12 ± 0.08 | 19.79 ± 0.08 | 9.18 ± 0.07 | 0.61 ± 0.07 | |
LGOX | l-Glu | 2.65 ± 0.11 | 2.93 ± 0.06 | 3.38 ± 0.05 | 1.28 ± 0.07 |
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Jing, X.-R.; Liu, H.; Nie, Y.; Xu, Y. 2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis. Int. J. Mol. Sci. 2020, 21, 5347. https://doi.org/10.3390/ijms21155347
Jing X-R, Liu H, Nie Y, Xu Y. 2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis. International Journal of Molecular Sciences. 2020; 21(15):5347. https://doi.org/10.3390/ijms21155347
Chicago/Turabian StyleJing, Xiao-Ran, Huan Liu, Yao Nie, and Yan Xu. 2020. "2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C–H Bond Oxidation for (2s,3r,4s)-4-Hydroxyisoleucine Synthesis" International Journal of Molecular Sciences 21, no. 15: 5347. https://doi.org/10.3390/ijms21155347