Isocitrate dehydrogenase from extremophiles; Molecular adaptations to high temperatures

Abstract

Life on earth has adapted to a wide variety of environmental conditions, many of which are extreme to us humans. Temperature is one of the most important factors limiting biological activity and survival, but so far, biological activity has been observed in a wide temperature range; from -20 to + 121 °C. The organisms living at different temperature ranges are classified according to their optimal growth temperature; psychrophiles below 15 °C, mesophiles 15-45 °C, thermophiles 45-80 °C and hyperthermophiles above 80 °C. The forces governing the high thermal stability of enzymes from (hyper)thermophiles are of great interest due to their possible applications in different industries. Although several comparative structural studies on homologous enzymes from mesophilic and (hyper)thermophilic organisms have been performed no universal feature responsible for the high thermal stability of (hyper)thermophilic enzymes have been observed. In order to understand more closely the molecular mechanisms underlaying protein stability, heat-adaptive features of isocitrate dehydrogenase (IDH) from two hyperthermophiles, Aeropyrum pernix (ApIDH) and Archaeoglobus fulgidus (AfIDH), have been analysed based on their 3D-structures and comparisons to mesophilic homologs, in particular to IDH from E. coli (EcIDH). Isocitrate dehydrogenase, a key enzyme in the tricarboxylic acid cycle (TCA), catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate using NAD(P)+ as cofactor. A previous study revealed ApIDH as a hyperstable IDH with an apparent melting temperature (Tm) of 109.9 °C, whereas AfIDH was less stable with a Tm of 98.5 °C. In the present work, Tms of the thermophilic Thermoplasma acidophilum IDH (TaIDH), Methylococcus capsulatus IDH (McIDH) and the mesophilic EcIDH were determined to 80.0, 70.3 and 52.6 °C, respectively. Each of the (hyper)thermophilic enzymes showed significantly higher Tm than EcIDH. To investigate the importance of certain structural traits for the thermal stability of the hyperthermophilic ApIDH and AfIDH, a mutational analysis and a domain-swapping experiment was performed, respectively. The hyperthermophilic IDHs showed additional stabilization of their N-terminus; ApIDH by a disulfide bond, and AfIDH through an aromatic cluster. The size and positioning of ionic networks differed among the hyperthermophilic ApIDH and AfIDH and the mesophilic EcIDH. ApIDH possessed a higher number of intra-and inter-subunit ion pairs and the ionic networks were larger than observed in AfIDH and EcIDH. Mutational disruption of a 7-membered inter-domain network demonstrated the importance of this ionic network in the thermal stability of the former enzyme. The hyperthermophilic AfIDH was, however, strikingly similar to EcIDH and possessed almost the same number of ion pairs and ionic networks. However, a unique inter-subunit 4-membered ionic network between the claspdomain and the small domain was found in the former enzyme. To investigate the contribution of the clasp-domain to the thermal stability of AfIDH, chimeras of the two enzymes were constructed by domain-swapping. An aromatic cluster which is believed to further strengthen the subunit interaction was also identified in ApDIH and AfIDH. This cluster also seems to be conserved conserved in the moderately thermostable TaIDH. The biochemical characterization of McIDH identified the first homotetrameric NAD+- dependent form of a bacterial IDH with a high sequence similarity to homoisocitrate dehydrogenase and isopropylmalate dehydrogenase. In conclusion, the work presented in this thesis has increased our knowledge on how enzymes from (hyper)thermophiles can adapt to and resist thermal denaturation at high temperatures.