Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon
In: Microorganisms, Jg. 9 (2021-11-01), Heft 11, S. 2318
Online
academicJournal
Zugriff:
Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota, such as “Candidatus 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 (MhIDH) was recombinantly produced in Escherichia coli and purified to electrophoretic homogeneity for biochemical characterization. MhIDH showed optimal activity around pH 8 and appeared to be specific for NADP+ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and kcat-values of 38.48 ± 1.62 and 43.99 ± 1.46 s−1 resulting in kcat/KM-values of 725 ± 107.62 and 22.69 ± 2.15 mM−1 s−1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can likely be attributed to the presence of a proline residue in the NADP+ binding pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure.
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Biochemical Characterization of Recombinant Isocitrate Dehydrogenase and Its Putative Role in the Physiology of an Acidophilic Micrarchaeon
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Autor/in / Beteiligte Person: | Winkler, Dennis ; Gfrerer, Sabrina ; Gescher, Johannes |
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Zeitschrift: | Microorganisms, Jg. 9 (2021-11-01), Heft 11, S. 2318 |
Veröffentlichung: | MDPI AG, 2021 |
Medientyp: | academicJournal |
ISSN: | 2076-2607 (print) |
DOI: | 10.3390/microorganisms9112318 |
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