Comparison of Isocitrate Dehydrogenase from Three Hyperthermophiles Reveals Differences in Thermostability, Cofactor Specificity, Oligomeric State, and Phylogenetic Affiliation

Abstract

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...

Phosphorylation controls the functioning ofStaphylococcus aureusisocitrate dehydrogenase – favours biofilm formation

“…This cycle is largely regulated by the expression of isocitrate dehydrogenase (IDH) 1 . In bacteria, the activity of IDH is controlled through phosphorylation and dephosphorylation [2][3][4] .…”

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

“…Prokaryotic ICDH in this group has previously been considered a homodimeric and an NAD(P)-dependent enzyme. However, due to increasing reports of NAD-dependent ICDH in bacteria (Acidithiobacillus, Aquifex, Hydrogenobacter, Methylophilus, and Streptococcus) and archaea (Pyrococcus) and of homotetrameric ICDH in bacteria (Methylococcus and Thermotoga) (3,6,7,9,(13)(14)(15)(22)(23), prokaryotic oligomeric ICDH is now recognized as an enzyme with various oligomeric states and coenzyme specificities.…”

“…Phylogenetic analyses of prokaryotic oligomeric ICDH indicate that this enzyme does not comprise a single lineage but can be divided into many subfamilies (21)(22)(23). EcICDH is one of the best analyzed forms and belongs to a distinctive subfamily that also contains ICDH from archaea (Aeropyrum, Archaeoglobus, Caldococcus, and Pyrococcus) and Aquificales (Aquifex and Hydrogenobacter) (3)(4)(5)(6)(21)(22)(23).…”

“…EcICDH is one of the best analyzed forms and belongs to a distinctive subfamily that also contains ICDH from archaea (Aeropyrum, Archaeoglobus, Caldococcus, and Pyrococcus) and Aquificales (Aquifex and Hydrogenobacter) (3)(4)(5)(6)(21)(22)(23). These enzymes can be considered a single lineage and can be categorized as EcICDH-type enzymes.…”

Supramolecular Complex Formation and Crystallization of Isocitrate Dehydrogenase fromThermus thermophilusHB8: Preliminary Studies with X-Ray Crystallography and Atomic Force Microscopy

“…ICDHs have been classified into three subfamilies based on sequence comparisons. 1,2) All archaeal and most bacterial ICDHs are classified together into subfamily I, eukaryotic homodimeric ICDHs and some bacterial ICDHs into subfamily II, and eukaryotic hetero-oligomeric ICDHs into subfamily III. In contrast with these homologous proteins, another type of NADP þ -dependent monomeric ICDHs, with molecular masses of 80-100 kDa, have been found.…”