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...
Guardado en:
| Autores principales: | , , |
|---|---|
| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
MDPI AG
2021
|
| Materias: | |
| Acceso en línea: | https://doaj.org/article/3d58cb9bb3ba4df48f84d57555fb0164 |
| Etiquetas: |
Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
|
| id |
oai:doaj.org-article:3d58cb9bb3ba4df48f84d57555fb0164 |
|---|---|
| record_format |
dspace |
| spelling |
oai:doaj.org-article:3d58cb9bb3ba4df48f84d57555fb01642021-11-25T18:25:05ZBiochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon10.3390/microorganisms91123182076-2607https://doaj.org/article/3d58cb9bb3ba4df48f84d57555fb01642021-11-01T00:00:00Zhttps://www.mdpi.com/2076-2607/9/11/2318https://doaj.org/toc/2076-2607Despite 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.Dennis WinklerSabrina GfrererJohannes GescherMDPI AGarticleacidophilesarchaeaMicrarchaeotaisocitrate dehydrogenaseBiology (General)QH301-705.5ENMicroorganisms, Vol 9, Iss 2318, p 2318 (2021) |
| institution |
DOAJ |
| collection |
DOAJ |
| language |
EN |
| topic |
acidophiles archaea Micrarchaeota isocitrate dehydrogenase Biology (General) QH301-705.5 |
| spellingShingle |
acidophiles archaea Micrarchaeota isocitrate dehydrogenase Biology (General) QH301-705.5 Dennis Winkler Sabrina Gfrerer Johannes Gescher Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon |
| 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. |
| format |
article |
| author |
Dennis Winkler Sabrina Gfrerer Johannes Gescher |
| author_facet |
Dennis Winkler Sabrina Gfrerer Johannes Gescher |
| author_sort |
Dennis Winkler |
| title |
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_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_sort |
biochemical characterization of recombinant isocitrate dehydrogenase and its putative role in the physiology of an acidophilic micrarchaeon |
| publisher |
MDPI AG |
| publishDate |
2021 |
| url |
https://doaj.org/article/3d58cb9bb3ba4df48f84d57555fb0164 |
| work_keys_str_mv |
AT denniswinkler biochemicalcharacterizationofrecombinantisocitratedehydrogenaseanditsputativeroleinthephysiologyofanacidophilicmicrarchaeon AT sabrinagfrerer biochemicalcharacterizationofrecombinantisocitratedehydrogenaseanditsputativeroleinthephysiologyofanacidophilicmicrarchaeon AT johannesgescher biochemicalcharacterizationofrecombinantisocitratedehydrogenaseanditsputativeroleinthephysiologyofanacidophilicmicrarchaeon |
| _version_ |
1718411188950269952 |