Winkler, DennisDennisWinklerGfrerer, SabrinaSabrinaGfrererGescher, JohannesJohannesGescher2021-11-172021-11-172021-11-09Microorganisms 9 (11): 2318 (2021)http://hdl.handle.net/11420/10958Despite 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⁺ yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed <i>K_M</i>-values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and <i>k_cat</i>-values of 38.48 ± 1.62 and 43.99 ± 1.46 s⁻¹ resulting in <i>k_cat</i>/<i>K_M</i>-values of 725 ± 107.62 and 22.69 ± 2.15 mM⁻¹ s⁻¹ for DL-isocitrate and NADP⁺, respectively. <i>Mh</i>IDH’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.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.en2076-2607202111Multidisciplinary Digital Publishing Institutehttps://creativecommons.org/licenses/by/4.0/acidophilesarchaeaMicrarchaeotaisocitrate dehydrogenaseBiowissenschaften, BiologieJournal Article2021-11-1110.15480/882.390910.3390/microorganisms911231810.15480/882.3909Poli, AnnaritaAnnaritaPoliFinore, IlariaIlariaFinoreOther