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Biochemical characterization of recombinant isocitrate dehydrogenase and its putative role in the physiology of an acidophilic micrarchaeon

Winkler, D.; Gfrerer, S.; Gescher, J.


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 K$_{M}$-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and k$_{cat}$-values of 38.48 ± 1.62 and 43.99 ± 1.46 s$^{-1}$ resulting in k$_{cat}$/K$_{M}$-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.

Verlagsausgabe §
DOI: 10.5445/IR/1000140695
Veröffentlicht am 15.12.2021
DOI: 10.3390/microorganisms9112318
Zitationen: 1
Zitationen: 1
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Angewandte Biowissenschaften (IAB)
Institut für Biologische Grenzflächen (IBG)
Publikationstyp Zeitschriftenaufsatz
Publikationsdatum 09.11.2021
Sprache Englisch
Identifikator ISSN: 2076-2607
KITopen-ID: 1000140695
HGF-Programm 43.33.11 (POF IV, LK 01) Adaptive and Bioinstructive Materials Systems
Erschienen in Microorganisms
Verlag MDPI
Band 9
Heft 11
Seiten Art.Nr. 2318
Bemerkung zur Veröffentlichung Gefördert durch den KIT-Publikationsfonds
Schlagwörter acidophiles; archaea; Micrarchaeota; isocitrate dehydrogenase
Nachgewiesen in Scopus
Web of Science
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