Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration
Abstract
:1. Introduction
2. Results
2.1. Expression and Purification of CgIDH and AvIDH
2.2. Quaternary Structure Analyses of CgIDH and AvIDH
2.3. Kinetic Parameters and Biochemical Properties of CgIDH and AvIDH
2.4. Regeneration of NADPH Using Monomeric CgIDH and AvIDH
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Plasmids, and Cloning
4.2. Protein Expression and Purification
4.3. Size Exclusion Chromatography
4.4. Enzyme Assay and Kinetic Parameters
4.5. Coupling Reactions of IDH with NADPH-Dependent P450 BM3
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Enzymes | NADP+ | Isocitrate | Ref. | ||||
---|---|---|---|---|---|---|---|
Km (μM) * | kcat (s−1) | kcat/Km (μM−1s−1) | Km (μM) | kcat (s−1) | kcat/Km (μM−1s−1) | ||
Previously reported CgIDH | 4.0 | 87.0 | 21.8 | 5.0 | 87.0 | 17.4 | [25] |
Previously reported AvIDH | 5.8 | 92.6 | 15.9 | 7.9 | 73.0 | 9.3 | [26] |
CgIDH | 10.3 | 43.7 | 4.3 | 15.0 | 49.4 | 3.3 | This study |
AvIDH | 5.9 | 38.7 | 6.6 | 13.9 | 44.0 | 3.2 | This study |
Enzyme | Optimum Temperature (°C) | Optimum pH | |||||
CgIDH | 40 | 7.5 | |||||
AvIDH | 40 | 7.5 |
Metal Ions * | Relative Activity (%) | |
---|---|---|
CgIDH | AvIDH | |
EDTA | ND | ND |
Mn2+ | 100 ± 6.1 | 100 ± 6.0 |
Mg2+ | 43.7 ± 2.9 | 50.0 ± 3.5 |
Ca2+ | 2.1 ± 0.3 | 1.3 ± 0.2 |
Cu2+ | 2.6 ± 0.5 | 2.1 ± 0.2 |
Co2+ | 6.4 ± 0.9 | 7.8 ± 0.3 |
Ni2+ | 2.7 ± 0.2 | 2.2 ± 0.1 |
Zn2+ | 8.8 ± 0.8 | 11.7 ± 0.1 |
Names | Content & Sequence | RE * |
---|---|---|
Plasmids | ||
pET24a | Kanr, T7 promoter, lacO, PBR322 ori | |
pET24a-CgIDH | idh gene from C. glutamicum subcloned into pET24a | |
pET24a-AvIDH | idh gene from A. vinelandii subcloned into pET24a | |
pET24a-LmG6PDH | g6pdh gene from L. mesenteroides subcloned into pET24a | |
pET24a-BM3 | CYP102A1 gene from B. megaterium subcloned into pET24a | |
Primers | ||
CgIDH F | 5′-GAAGGAGATATACATATGGCTAAGATCATCTGGACCCG-3′ | NdeI |
CgIDH R | 5′-GTGGTGGTGGTGCTCGAGCTTCTTCAGTGCGTCAACGATCTC-3′ | XhoI |
AvIDH F | 5′-GAAGGAGATATACATATGTCCACACCGAAGATTATCTATACGC-3′ | NdeI |
AvIDH R | 5′-GTGGTGGTGGTGCTCGAGTGCAAGAGGTGCCAGAGCC-3′ | XhoI |
LmG6PDH F | 5′-TAAGAAGGAGATATACATATGGTTTCAGAAATCAAGACGTTAGTAACTTTC-3′ | NdeI |
LmG6PDH R | 5′-GTGGTGGTGGTGCTCGAGACCTTTAAACACCCAAGCATCACC-3′ | XhoI |
BM3 F | 5′-TAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGA GATATACATATGAC-3′ | XbaI |
BM3 R | 5′-GCTGCCTCCTTTGCTGCTGCCTCCTTAGCAGCAGCTTCCCCAGCCC ACACGTCTTTTGC-3′ |
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Lee, H.-D.; Yoo, S.-K.; Yoo, H.-S.; Yun, C.-H.; Kim, G.-J. Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration. Int. J. Mol. Sci. 2022, 23, 15318. https://doi.org/10.3390/ijms232315318
Lee H-D, Yoo S-K, Yoo H-S, Yun C-H, Kim G-J. Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration. International Journal of Molecular Sciences. 2022; 23(23):15318. https://doi.org/10.3390/ijms232315318
Chicago/Turabian StyleLee, Hun-Dong, Su-Kyoung Yoo, Ho-Seok Yoo, Chul-Ho Yun, and Geun-Joong Kim. 2022. "Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration" International Journal of Molecular Sciences 23, no. 23: 15318. https://doi.org/10.3390/ijms232315318