The crystal structure of a hyperthermostable subfamily II isocitrate dehydrogenase from Thermotoga maritima

Abstract

Isocitrate dehydrogenase (IDH) from the hyperthermophile Thermotoga maritima (TmIDH) catalyses NADP+‐ and metal‐dependent oxidative decarboxylation of isocitrate to α‐ketoglutarate. It belongs to the β‐decarboxylating dehydrogenase family and is the only hyperthermostable IDH identified within subfamily II. Furthermore, it is the only IDH that has been characterized as both dimeric and tetrameric in solution. We solved the crystal structure of the dimeric apo form of TmIDH at 2.2 Å. The R‐factor of the refined model was 18.5% (Rfree 22.4%). The conformation of the TmIDH structure was open and showed a domain rotation of 25–30° compared with closed IDHs. The separate domains were found to be homologous to those of the mesophilic mammalian IDHs of subfamily II and were subjected to a comparative analysis in order to find differences that could explain the large difference in thermostability. Mutational studies revealed that stabilization of the N‐ and C‐termini via long‐range electrostatic interactions were important for the higher thermostability of TmIDH. Moreover, the number of intra‐ and intersubunit ion pairs was higher and the ionic networks were larger compared with the mesophilic IDHs. Other factors likely to confer higher stability in TmIDH were a less hydrophobic and more charged accessible surface, a more hydrophobic subunit interface, more hydrogen bonds per residue and a few loop deletions. The residues responsible for the binding of isocitrate and NADP+ were found to be highly conserved between TmIDH and the mammalian IDHs and it is likely that the reaction mechanism is the same.

Defining the Role of Salt Bridges in Protein Stability

“…Recently, an increasing number of observations strengthens the view, that electrostatic interactions are an important factor conferring thermostability to proteins, although opposite views also exist (89)(90)(91)(92)(93)(94). The overall trend of an increased number of salt bridges in proteins from organisms with higher optimal growth temperature is illustrated in Fig.…”

Structural and Functional Properties of Isocitrate Dehydrogenase from the Psychrophilic Bacterium Desulfotalea psychrophila Reveal a Cold-active Enzyme with an Unusual High Thermal Stability

“…13 The level of primary sequence identity is low between IDHs from subfamily I and II, but their 3D structures are rather conserved, as are the residues involved in isocitrate binding and catalysis. 8,12 The most pronounced differences between subfamily I and subfamily II are the structural architecture of the so-called clasp domain, which involves residues from each of the two subunits, locking the subunits together. The other major difference is the distance between the N and C termini, which are separated by 4 Å in subfamily II IDHs (e.g.…”

“…9 Recently, we solved the structure of the hyperthermophilic IDHs from Aeropyrum pernix (ApIDH, PDB codes 1XGV, 1TYO and 1XKD), 10 Archaeoglobus fulgidus (AfIDH, PDB 2IV09) 11 and Thermotoga maritima (TmIDH, PDB 1ZOR). 12 The IDH enzyme family is very diverse with respect to cofactor specificity and oligomeric states, and is divided into three different subfamilies on the basis of primary structure where EcIDH, BsIDH, ApIDH, AfIDH belong to subfamily I and PcIDH, HcIDH and TmIDH belong to subfamily II, respectively. 13 The level of primary sequence identity is low between IDHs from subfamily I and II, but their 3D structures are rather conserved, as are the residues involved in isocitrate binding and catalysis.…”

Stabilizing Ionic Interactions in a Full-consensus Ankyrin Repeat Protein

“…Similar results were found in another comprehensive structural bioinformatics study 26 and in more specialized studies on individual protein families. [27][28][29][30] The high number of salt bridges in hyperthermophilic proteins was explained by the diminishing desolvation penalty for salt bridges at increasing temperatures. 31 Therefore, the contribution of salt bridges to protein stability becomes more important at higher temperatures.…”