TY - JOUR T1 - Acidophilic Micrarchaeon Seems to Maintain a Slightly Alkaline Cytosolic pH JF - bioRxiv DO - 10.1101/2021.05.02.442328 SP - 2021.05.02.442328 AU - Dennis Winkler AU - Sabrina Gfrerer AU - Johannes Gescher Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/05/02/2021.05.02.442328.abstract N2 - Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. “Candidatus Micrarchaeum harzensis A_DKE”, for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for bio-chemical characterisation. MhIDH 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 s-1 and 43.99±1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely 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. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.Competing Interest StatementThe authors have declared no competing interest. ER -