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 " 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 (IDH) was recombinantly produced in and purified to electrophoretic homogeneity for biochemical characterization. IDH showed optimal activity around pH 8 and appeared to be specific for NADP yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed -values of 53.03 ± 5.63 µM and 1.94 ± 0.12 mM and -values of 38.48 ± 1.62 and 43.99 ± 1.46 s resulting in /-values of 725 ± 107.62 and 22.69 ± 2.15 mM s for DL-isocitrate and NADP, respectively. 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.