Contrasting effects of selenite and tellurite on lipoamide dehydrogenase activity suggest a different biological behaviour of the two chalcogens

The effects of selenite and tellurite on the mammalian enzyme lipoamide dehydrogenase were compared. Selenite acts as a substrate of lipoamide dehydrogenase in a process requiring the presence of lipoamide. In contrast, tellurite is a potent inhibitor, effective in the low micromolar range. The inhibitory effect of tellurite on lipoamide dehydrogenase is partially reverted by dithiothreitol indicating the participation of the thiol groups of the enzyme. Tellurite, but not selenite, stimulates the diaphorase activity of lipoamide dehydrogenase. In a mitochondrial matrix protein preparation, which contains lipoamide dehydrogenase, an inhibitory action similar to that observed on the purified enzyme was also elicited by tellurite. Human embryonic kidney cells (HEK 293 T) treated with tellurite show a partial inhibition of lipoamide dehydrogenase. In addition to the toxicological implications of tellurium compounds, the reported results suggest that tellurite and its derivatives can be used as potential tools for studying biochemical reactions.     

pH-dependent substrate preference of pig heart lipoamide dehydrogenase varies with oligomeric state: response to mitochondrial matrix acidification

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of LADH entrapped in reverse micelles reveals dimer and tetramer at both pH 5.8 and 7.5, but monomer was observed only at pH 5.8. Enzyme activity measurements in reverse Aerosol OT micelles in octane indicate that LADH dimer and tetramer possess lipoamide dehydrogenase and diaphorase activities at pH 7.5. Upon acidification to pH 5.8 only the LADH monomer is active and only the diaphorase activity is observed. These results indicate a correlation between pH-dependent changes in the LADH reaction specificity and its oligomeric state. The acidification of mitochondrial matrix that occurs during ischemia-reperfusion injury is sufficient to alter the structure and enzymatic specificity of LADH, thereby reducing mitochondrial defenses, increasing oxidative stress, and slowing the recovery of energy metabolism. Matrix acidification may also disrupt the quaternary structure of other mitochondrial protein complexes critical for cellular homeostasis and survival.     

On the FAD-induced dimerization of apo-lipoamide dehydrogenase from Azotobacter vinelandii and Pseudomonas fluorescens. Kinetics of reconstitution

The apoenzymes of lipoamide dehydrogenase from pig heart and from Pseudomonas fluorescens were prepared at pH 2.7 and pH 4.0, respectively, using a hydrophobic interaction chromatography procedure recently developed for lipoamide dehydrogenase from Azotobacter vinelandii and other flavoproteins [Van Berkel et al. (1988) Eur. J. Biochem. 178, 197-207]. The apoenzyme from pig heart, having 5% of residual activity, shows an equilibrium between the monomeric and dimeric species. Both the yield and the degree of reconstitution of dimeric holoenzyme is 75% of starting material under optimal conditions. The kinetics of reconstitution of pig heart apoenzyme differ slightly from that obtained with the apoenzyme prepared by acid ammonium sulfate precipitation at pH 1.5 [Kalse, J. F. and Veeger, C. (1968) Biochim. Biophys. Acta 159, 244-256]. The apoenzyme from P. fluorescens is in the monomeric state and shows negligible residual activity. The yield and degree of reconstitution of the dimeric holoenzyme is more than 90% of starting material. Reconstitution of the apoenzymes from A. vinelandii and P. fluorescens involves minimally a two-step sequential process. Initial flavin-binding results in regaining of full dichloroindophenol activity, quenching of tryptophan fluorescence and strong increase of FAD fluorescence polarization. In the second step, dimerization occurs as reflected by regain of lipoamide activity, strongly increased FAD fluorescence and increased hyperchroism of the visible absorption spectrum. The kinetics of FAD-induced dimerization are strongly dependent on the apoenzyme used. At 0 degrees C, the monomeric apoenzyme-FAD complex is either stabilized (P. fluorescens) or only transiently detectable (A. vinelandii). Dimerization of P. fluorescens enzyme is strongly stimulated in the presence of NADH.     

The conformational stability of the redox states of lipoamide dehydrogenase from Azotobacter vinelandii.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotobacter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, tm = 80 degrees C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyme or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass greater than 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu459 and/or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.     

Lipoamide dehydrogenase from Malbranchea pulchella: isolation and characterization.

Lipoamide dehydrogenase (EC 1.6.4.3) has been isolated from a total homogenate of frozen mycelium of the thermophilic fungus Malbranchea pulchella var. sulfurea by a three-step procedure involving ammonium sulfate fractionation, Procion Brilliant Blue M-R--Sepharose 4B chromatography, and hydroxylapatite chromatography. The second step is the key purification step with the Procion Brilliant Blue M-R dye acting as an affinity ligand for the enzyme. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme is a dimer of molecular weight 102 000, and each monomer of 51 000 molecular weight binds one molecule of flavin adenine dinucleotide. Other properties determined include a pH optimum of 8.2, a strong specificity for the substrates dihydrolipoamide and nicotinamide adenine dinucleotide, the apparent lack of multiple enzymic forms, the presence of diaphorase activity, and resistance to temperature denaturation up to 60 degrees C. The amino acid composition and absorption spectrum of the enzyme were also determined. The properties of lipoamide dehydrogenase from this source are very similar to those reported for the enzyme from serveral other sources.     

Interaction of lipoamide dehydrogenase with the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii

The interaction between lipoamide dehydrogenase (E3) and dihydrolipoyl transacetylase (E2p) from the pyruvate dehydrogenase complex was studied during the reconstitution of monomeric E3 apoenzymes from Azotobacter vinelandii and Pseudomonas fluorescens. The dimeric form of E3 is not only essential for catalysis but also for binding to the E2p core, because the apoenzymes as well as a monomeric holoenzyme from P. fluorescens, which can be stabilized as an intermediate at 0 degree C, do not bind to E2p. Lipoamide dehydrogenase from A. vinelandii contains a C-terminal extension of 15 amino acids with respect to glutathione reductase which is, in contrast to E3, presumably not part of a multienzyme complex. Furthermore, the last 10 amino acid residues of E3 are not visible in the electron density map of the crystal structure and are probably disordered. Therefore, the C-terminal tail of E3 might be an attractive candidate for a binding region. To probe this hypothesis, a set of deletions of this part was prepared by site-directed mutagenesis. Deletion of the last five amino acid residues did not result in significant changes. A further deletion of four amino acid residues resulted in a decrease of lipoamide activity to 5% of wild type, but the binding to E2p was unaffected. Therefore it is concluded that the C-terminus is not directly involved in binding to the E2p core. Deletion of the last 14 amino acids produced an enzyme with a high tendency to dissociate (Kd approximately 2.5 microM). This mutant binds only weakly to E2p. The diaphorase activity was still high. This indicates, together with the decreased Km for NADH, that the structure of the monomer is not appreciably changed by the mutation. Rather the orientation of the monomers with respect to each other is changed. It can be concluded that the binding region of E3 for E2p is constituted from structural parts of both monomers and binding occurs only when dimerization is complete.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Multifunctionality of lipoamide dehydrogenase: Promotion of electron transferase reaction

Various approaches to promote the one-electron transfer reaction of lipoamide dehydrogenase have been investigated. An addition of riboflavin facilitates the electron transfer between NADH and Fe(CN)₆^3?. Aminocarboxymethylation and cadmium derivatization of the catalytic disulfide moderately activate the electron transfer reaction. An enhancement in the electron transferase activity of the Co(II)-enzyme complex is associated with decreased Michaelis and inhibition constants. Phosphopyridoxamidation identifies the suppressive effect on the electron transferase activity of carboxyl groups proximal to the catalytic histidine residue of lipoamide dehydrogenase. Amidation of these carboxyl groups with diamine greatly promote the one-electron transfer reaction. The increased electron transferase activity of the amidated enzyme is related to the charge nature of the amidated nucleophile and associated with the increased catalytic efficiency which undergoes a shift in the pH profile. The introduction of cationic aminoethyl groups presumably encourages the formation of an anionic flavosemiquinone which promotes the one-electron transfer reaction.     

Purification of lipoamide dehydrogenase from Ascaris muscle mitochondria and its relationship to NADH:NAD+ transhydrogenase activity

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3) has been isolated from Ascaris suum muscle mitochondria. This activity has been purified to apparent homogeneity from both the pyruvate dehydrogenase complex and from 150,000g mitochondrial supernatants which were devoid of pyruvate dehydrogenase complex activity. The enzymes from both sources exhibited similar kinetic, catalytic, and regulatory properties and appear to be identical as judged by polyacrylamide gel electrophoresis. The native enzyme acts as a dimer, containing 2 mol of FAD, and has a subunit molecular weight of 54,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel chromatography. The enzyme also possesses substantial NADH:NAD⁺ transhydrogenase activity. Heat denaturation and differential solubilization experiments imply that the transhydrogenase activity previously reported is, in fact, associated with the lipoamide dehydrogenase moiety of the Ascaris pyruvate dehydrogenase complex. Whether or not this activity functions physiologically in hydride ion translocation, as previously suggested, remains to be demonstrated.     

Inactivation of Lipoamide Dehydrogenase by Cobalt(II) and Iron(II) Fenton Systems: Effect of Metal Chelators, Thiol Compounds and Adenine Nucleotides

Fe(II)-and Co(II)-Fenton systems (FS) inactivated the lipoamide reductase activity but not the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). The Co(II) system was the more effective as LADH inhibitor. Phosphate ions enhanced the Fe(II)-FS activity. EDTA, DETAPAC, DL-histidine, DL-cysteine, glutathione, DL-dithiothreitol, DL-lipoamide, DL-thioctic acid, bathophenthroline, trypanothione and ATP, but not ADP or AMP, prevented LADH inactivation. Reduced disulfide compounds were more effective protectors than the parent compounds. Mg ions counteracted ATP protective action. Glutathione and DL-dithiothreitol partially restored the lipoamide dehydrogenase activity of the Fe(II)-FS-inhibited LADH. DL-histidine exerted a similar action on the Co(II)-FS-inhibited enzyme. Ethanol, mannitol and benzoate did not prevent LADH inactivation by the assayed Fenton systems and, accordingly, it is postulated that site-specific generated HO'radicals were responsible for LADH inactivation. With the Co(II)-FS, oxygen reactive species other than HO, might contribute to LADH inactivation.     

Lipoamide dehydrogenase from Trypanosoma cruzi: some properties and cellular localization.

Lipoamide dehydrogenase (E.C. 1.6.4.3) was found in Trypanosoma cruzi, Tulahuen strain, stocks Tul-2 and Q501, and CA-1 strain. After differential centrifugation of epimastigote homogenates, ammonium sulfate fractionation of the 105,000 g supernatant yielded a partially purified preparation which precipitated between 0.40 and 0.80 ammonium sulfate saturation. The enzyme (a) catalyzed the oxidation of dihydrolipoamide by NAD+ and the reduction of lipoamide by NADH, the forward reaction being 2.5-fold faster than the reverse reaction; (b) exhibited hyperbolic dependence on substrate concentration and (c) possessed diaphorase activity which was less than 5% of the lipoamide reductase activity. The NADH-reduced enzyme was inhibited by arsenite, cadmium and p-chloromercuribenzoate in a concentration-dependent manner. Substrate specificity allowed lipoamide dehydrogenase to be differentiated from T. cruzi trypanothione reductase and other NADPH-dependent flavoenzymes. After cell disruption, lipoamide dehydrogenase was found mostly in the cytosolic fraction and no evidence for association with the plasma membrane was obtained.     

Kinetic and spectroscopic studies of transhydrogenase activity and nucleotide site of lipoamide dehydrogenase

The kinetic behavior and spectroscopic characteristics of the nucleotide site(s) of lipoamide dehydrogenase have been investigated. Both subunits of the dimeric enzyme interact with NAD+. The binding of NAD+ is associated with a negative trough around 420-450 nm and a positive peak at 507 nm of the difference spectrum. The transhydrogenation between NADH and thionicotinamide nucleotide or acetylpyridine nucleotide is shown to proceed via a Ping Pong or an ordered Bi Bi mechanism, respectively, at pH above 7.0. Lowering pH or acetamidation lose the spectral characteristic of the positive peak of the enzyme-NAD+ complex with a concurrent change in the kinetic mechanism in the NADH+-acetylpyridine nucleotide transhydrogenation.     

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.     

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.     

The use of pig heart dihydrolipoamide dehydrogenase (diaphorase) for the regeneration of NADH or NAD

Summary Incubations of freshly dissolved diaphorase with reduced methylviologen show in the first hours variable but usually rather low activities for the reduction of NAD with reduced methylviologen, as compared to lipoamide dehydrogenase activity at the expense of NADH. However, the former activity increases in a few hours by factors of 5–10 and is then very stable under operational conditions. The half life in the presence of reduced methylviologen at 35°C is >40 days, whereas the lipoamide dehydrogenase activity has a half life of only about 4 h. Even in a rigorously stirred electro-chemical cell the methylviologen dependent NAD reductase activity is very stable.The enzyme is also suitable for the regeneration of NAD if carbamoylmethylviologen is used. This mediator has a 145 mV less negative redox potential than methylviologen. Again the stability of the enzyme is rather high. Under operational conditions the activity increases for about 9 days and then stays constant for at least 11 days.     

Malic acid production by an electrochemical reduction system combined with the use of diaphorase and methylviologen

A regenerating reaction combined with the use of native malate dehydrogenase, native diaphorase, methylviologen, NAD, oxalacetic acid as the substrate and lipoamide as a stabilizer was carried out in the presence of electrolysis. Consequently, malic acid was efficiently produced from oxalacetic acid in the regenerating reaction. A glassy carbon bead electrode was used as a cathode. Twenty four milliamperes were passed at a rotation speed of 500 rpm, 29.8 +/- 0.3 degrees C and -1.0 V. It was found that lipoamide has a stabilizing effect on malate dehydrogenase and diaphorase. Low concentration (50 muM) of NAD was also effective for the stabilization of malate dehydrogenase. NADH regeneration activity based on malic acid production rate was 4.7 U/mg of the enzyme protein of the commercial diaphorase preparation. The current efficiency was more than 74%, compared with the theoretical yield, in the presence of enough oxalacetic acid.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

A highly specific monomeric isocitrate dehydrogenase from Corynebacterium glutamicum

The monomeric isocitrate dehydrogenase (IDH) of Corynebacterium glutamicum is compared to the topologically distinct dimeric IDH of Escherichia coli. Both IDHs have evolved to efficiently catalyze identical reactions with similar pH optimum as well as striking specificity toward NADP and isocitrate. However, the monomeric IDH is 10-fold more active (calculated as kcat/Km.isocitrate/Km.NADP) and 7-fold more NADP-specific than the dimeric enzyme, favoring NADP over NAD by a factor of 50,000. Such an extraordinary coenzyme specificity is not rivaled by any other characterized dehydrogenases. In addition, the monomeric enzyme is 10-fold more specific for isocitrate. The spectacular substrate specificity may be predominantly attributed to the isocitrate-assisted stabilization of catalytic complex during hydride transfer. No significant overall sequence identity is found between the monomeric and dimeric enzymes. However, structure-based alignment leads to the identification of three regions in the monomeric enzyme that match closely the three motifs located in the central region of dimeric IDHs and the homologous isopropylmalate dehydrogenases. The role of Lys253 as catalytic residue has been demonstrated by site-directed mutagenesis. Our results suggest that monomeric and dimeric forms of IDHs are functionally and structurally homologous.