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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| Autores principales: | , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/3d58cb9bb3ba4df48f84d57555fb0164 |
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| Sumario: | 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. |
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