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 “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 _Mand 1.94 _ 0.12mMand kcat-values of 38.48 _ 1.62 and 43.99 _ 1.46 s1 resulting in kcat/KM-values of 725 _ 107.62 and 22.69 _ 2.15 mM1 s1 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.

Subjects

acidophiles

archaea

Micrarchaeota

isocitrate dehydrogenase

DDC Class

570: Biowissenschaften, Biologie

Publication version

publishedVersion

Lizenz

https://creativecommons.org/licenses/by/4.0/

Name

microorganisms-09-02318-v2.pdf

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3.18 MB

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Adobe PDF

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