Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon
Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “<i>Candidatus</i> Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is diff...
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| Format: | Article |
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MDPI AG
2021-11-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/9/11/2318 |
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| author | Dennis Winkler Sabrina Gfrerer Johannes Gescher |
| author_facet | Dennis Winkler Sabrina Gfrerer Johannes Gescher |
| author_sort | Dennis Winkler |
| collection | DOAJ |
| description | Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “<i>Candidatus</i> Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (<i>Mh</i>IDH) was recombinantly produced in <i>Escherichia coli</i> and purified to electrophoretic homogeneity for biochemical characterization. <i>Mh</i>IDH showed optimal activity around pH 8 and appeared to be specific for NADP<sup>+</sup> yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed <i>K<sub>M</sub></i>-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and <i>k<sub>cat</sub></i>-values of 38.48 ± 1.62 and 43.99 ± 1.46 s<sup>−1</sup> resulting in <i>k<sub>cat</sub></i>/<i>K<sub>M</sub></i>-values of 725 ± 107.62 and 22.69 ± 2.15 mM<sup>−1</sup> s<sup>−1</sup> for DL-isocitrate and NADP<sup>+</sup>, respectively. <i>Mh</i>IDH’s exceptionally low affinity for NADP<sup>+</sup>, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP<sup>+</sup> binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. |
| first_indexed | 2024-03-10T05:14:36Z |
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| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T05:14:36Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
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| series | Microorganisms |
| spelling | doaj.art-3d58cb9bb3ba4df48f84d57555fb01642023-11-23T00:29:37ZengMDPI AGMicroorganisms2076-26072021-11-01911231810.3390/microorganisms9112318Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic MicrarchaeonDennis Winkler0Sabrina Gfrerer1Johannes Gescher2Department of Applied Biology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131 Karlsruhe, GermanyDepartment of Applied Biology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131 Karlsruhe, GermanyDepartment of Applied Biology, Institute for Applied Biosciences, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 2, 76131 Karlsruhe, GermanyDespite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “<i>Candidatus</i> Micrarchaeum harzensis A_DKE”, remains largely enigmatic, as they highly express numerous genes encoding hypothetical proteins. Due to a lacking genetic system, it is difficult to elucidate the biological function of the corresponding proteins and heterologous expression is required. In order to prove the viability of this approach, A_DKE’s isocitrate dehydrogenase (<i>Mh</i>IDH) was recombinantly produced in <i>Escherichia coli</i> and purified to electrophoretic homogeneity for biochemical characterization. <i>Mh</i>IDH showed optimal activity around pH 8 and appeared to be specific for NADP<sup>+</sup> yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed <i>K<sub>M</sub></i>-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and <i>k<sub>cat</sub></i>-values of 38.48 ± 1.62 and 43.99 ± 1.46 s<sup>−1</sup> resulting in <i>k<sub>cat</sub></i>/<i>K<sub>M</sub></i>-values of 725 ± 107.62 and 22.69 ± 2.15 mM<sup>−1</sup> s<sup>−1</sup> for DL-isocitrate and NADP<sup>+</sup>, respectively. <i>Mh</i>IDH’s exceptionally low affinity for NADP<sup>+</sup>, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP<sup>+</sup> binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.https://www.mdpi.com/2076-2607/9/11/2318acidophilesarchaeaMicrarchaeotaisocitrate dehydrogenase |
| spellingShingle | Dennis Winkler Sabrina Gfrerer Johannes Gescher Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon Microorganisms acidophiles archaea Micrarchaeota isocitrate dehydrogenase |
| title | Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| title_full | Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| title_fullStr | Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| title_full_unstemmed | Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| title_short | Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| title_sort | biochemical characterization of recombinant isocitrate dehydrogenase and its putative role in the physiology of an acidophilic micrarchaeon |
| topic | acidophiles archaea Micrarchaeota isocitrate dehydrogenase |
| url | https://www.mdpi.com/2076-2607/9/11/2318 |
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