Structure determination, thermal stability and catalytic mechanism of hyperthermostable isocitrate dehydrogenases

Abstract

In this thesis, studies on the enzyme isocitrate dehydrogenase (IDH) from the hyperthermophiles Aeropyrum pernix (ApIDH), Thermotoga maritima (TmIDH), Archaeoglobus fulgidus (AfIDH) and Pyrococcus furiosus (PfIDH) are presented. The aim of the described work was to contribute to the understanding of protein thermostability and to study the catalytic mechanism.

Hyperthermophilic organisms thrive at temperatures ranging from WC to about 120 degress C. The three-dimensional structures of their enzymes are usually very similar to their counterparts in mesophilic organisms which optimally grow at 20-50 degrees C. Their similarity suggests that they are evolutionary related. However, the hyperthermostable enzymes must have specific properties which are distinct from those of the mesophilic homologues in order to avoid thermal denaturation. These properties are most likely determined by optimization of the weak forces by which proteins are stabilized.

IDH is an enzyme in the citric-acid cycle and is widely distributed in the three domains of life: Archaea, Bacteria and Eukarya. It catalyzes the metal-dependent dehydrogenation and decarboxylation of isocitrate to alpha-ketoglutarate and C02 using NAD+ or NADP+ as cofactor. It was found that ApIDH was most thermostable, with an apparent melting temperature of 109.9 degrees C, whereas that of TmIDH, AfIDH and PfIDH was 98.3 degrees C, 98.5 degrees C and 103.7 degrees C, respectively. The crystal structures of the apo-forms of ApIDH, TmIDH and AfIDH were determined. The structures indicated that the enzymes are homodimers and homologous to their mesophilic counterparts. In addition, the crystal structure of a ternary complex of ApIDH with NADP+, Ca2+ and isocitrate was determined. It was suggested that large domain movements and a proton relay chain might be essential for the catalytic reaction of IDH. The structures were compared to their mesophilic homologues and combined with mutational studies in order to reveal determinants for their increased thermostability.

The major findings were that ApIDH is stabilized by a disulfide bond at the N-terminus, a seven-membered ionic network and most likely by a higher total number of ion pairs; TmIDH is stabilized by long-range electrostatic interactions at the N- and C-termini, aromatic clustering, a higher total number of ion pairs and more inter-subunit ion pairs; AfIDH is stabilized through aromatic interactions at the Nterminus, inter-domain ionic networks and inter-domain aromatic clustering. In solution, ApIDH was found to be monomeric at pH 3.0 and dimeric at pH 7.5. Equilibrium unfolding characteristics and kinetic stability of ApIDH were investigated by spectroscopic methods. Although the ApIDH monomer contains three domains, the unfolding transition was reversible and could be approximated by a two-state process. The mutant R211M, which disrupts the seven-membered ionic network, was compared to the recombinant wildtype form. The apparent thermodynamic and kinetic stability of the mutant was significantly reduced and it was concluded that ionic networks contribute to the kinetic barriers that prevent unfolding of proteins from hyperthermophiles.