With the aim of gaining insight into the molecular and phylogenetic relationships of isocitrate dehydrogenase (IDH) from hyperthermophiles, we carried out a comparative study of putative IDHs identified in the genomes of the eubacterium Thermotoga maritima and the archaea Aeropyrum pernix and Pyrococcus furiosus. An optimum for activity at 90°C or above was found for each IDH. PfIDH and ApIDH were the most thermostable with a melting temperature of 103.7 and 109.9°C, respectively, compared with 98.3 and 98.5°C for TmIDH and AfIDH, respectively. Analytical ultracentrifugation revealed a tetrameric oligomeric state for TmIDH and a homodimeric state for ApIDH and PfIDH. TmIDH and ApIDH were NADP-dependent (K m(NADP) of 55.2 and 44.4 M, respectively) whereas PfIDH was NAD-dependent (K m(NAD) of 68.3 M). These data document that TmIDH represents a novel tetrameric NADP-dependent form of IDH and that PfIDH is a homodimeric NAD-dependent IDH not previously found among the archaea. The homodimeric NADP-IDH present in A. pernix is the most common form of IDH known so far. The evolutionary relationships of ApIDH, PfIDH, and TmIDH with all of the available amino acid sequences of di-and multimeric IDHs are described and discussed.Isocitrate dehydrogenases (IDHs) 1 are a broadly distributed and well characterized group of enzymes that catalyze the oxidative decarboxylation of D-isocitrate to 2-oxoglutarate and CO 2 with NAD ϩ (EC 1.1.1.41) or NADP ϩ (EC 1.1.1.42) as cofactor. IDH from Escherichia coli has been studied extensively with regard to its catalytic mechanism, kinetics, and regulation, and so far the three-dimensional structure is available only for IDH from E. coli and Bacillus subtilis (1-11). Resolution of the structure of NAD-dependent homodimeric isopropylmalate dehydrogenase (IMDH) from Thermus thermophilus revealed that this enzyme, together with EcIDH, represents a unique class of metal-dependent decarboxylating dehydrogenases that lack the ␤␣␤␣␤ motif characteristic of the nucleotide-binding Rossman fold (12, 13). IDH and IMDH are specific for structurally similar substrates of the form HOOC(OH)CHCH(X), in which X represents CH 2 COOH for IDH and CH(CH 3 ) 2 for IMDH (14, 15). However, EcIDH and T. thermophilus IMDH exhibit strong preference for their natural substrate, and the structural determinants for substrate and cofactor specificity have been identified (16 -20) Recently, tartrate dehydrogenase (TDH) and homoisocitrate dehydrogenase (HDH) have been suggested as members of the metal-dependent decarboxylating dehydrogenases (21-23).The IDHs comprise a diverse enzyme family with regard to cofactor specificity, primary structure, and oligomeric state. The archaea Archaeoglobus fulgidus, Caldococcus noboribetus, Haloferax volcanii, Sulfolobus solfataricus, and Sulfolobus acidocaldarius contain a single homodimeric IDH that is NADP-dependent or shows dual coenzyme specificity (24 -27). A homodimeric NAD-IDH is present in Methylophilus methylotrophus (28). However, most bacteria contain a single homodime...

Crystal and small-angle X-ray scattering structures of full-length human SUFU alone and in complex with the conserved SYGHL motif from GLI transcription factors show major conformational changes associated with binding and reveal an intrinsically disordered region crucial for pathway activation.

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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.

Isocitrate dehydrogenase from Archaeoglobus fulgidus (AfIDH) has an apparent melting temperature (T(m)) of 98.5 degrees C. To identify the structural features involved in thermal stabilization of AfIDH, the structure was solved to 2.5 A resolution. AfIDH was strikingly similar to mesophilic IDH from Escherichia coli (EcIDH) and displayed almost the same number of ion pairs and ionic networks. However, two unique inter-domain networks were present in AfIDH; one three-membered ionic network between the large and the small domain and one four-membered ionic network between the clasp and the small domain. The latter ionic network was presumably reduced in size when the clasp domain of AfIDH was swapped with that of EcIDH and the T (m) decreased by 18 degrees C. Contrarily, EcIDH was only stabilized by 4 degrees C by the clasp domain of AfIDH, a result probably due to the introduction of a unique inter-subunit aromatic cluster in AfIDH that may strengthen the dimeric interface in this enzyme. A unique aromatic cluster was identified close to the N-terminus of AfIDH that could provide additional stabilization of this region. Common and unique heat adaptive traits of AfIDH with those recently observed for hyperthermophilic IDH from Aeropyrum pernix (ApIDH) and Thermotoga maritima (TmIDH) are discussed herein.