Human NAD-dependent Isocitrate Dehydrogenase Research Articles

Dynamically driven correlations in elastic net models reveal sequence of events and causality in proteins.

An explicit analytic solution is given for the Langevin equation applied to the Gaussian Network Model of a protein subjected to both a random and a deterministic periodic force. Synchronous and asynchronous components of time correlation functions are derived and an expression for phase differences in the time correlations of residue pairs is obtained. The synchronous component enables the determination of dynamic communities within the protein structure. The asynchronous component reveals causality, where the time correlation function between residues i and j differs depending on whether i is observed before j or vice versa, resulting in directional information flow. Driver and driven residues in the allosteric process of cyclophilin A and human NAD-dependent isocitrate dehydrogenase are determined by a perturbation-scanning technique. Factors affecting phase differences between fluctuations of residues, such as network topology, connectivity, and residue centrality, are identified. Within the constraints of the isotropic Gaussian Network Model, our results show that asynchronicity increases with viscosity and distance between residues, decreases with increasing connectivity, and decreases with increasing levels of eigenvector centrality.

Structures of a constitutively active mutant of human IDH3 reveal new insights into the mechanisms of allosteric activation and the catalytic reaction

Human NAD-dependent isocitrate dehydrogenase or IDH3 (HsIDH3) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It consists of three types of subunits (α, β, and γ) and exists and functions as the (αβαγ)2 heterooctamer. HsIDH3 is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. Our previous studies have revealed the molecular basis for the activity and regulation of the αβ and αγ heterodimers. However, the molecular mechanism for the allosteric activation of the HsIDH3 holoenzyme remains elusive. In this work, we report the crystal structures of the αβ and αγ heterodimers and the (αβαγ)2 heterooctamer containing an α-Q139A mutation in the clasp domain, which renders all the heterodimers and the heterooctamer constitutively active in the absence of activators. Our structural analysis shows that the α-Q139A mutation alters the hydrogen-bonding network at the heterodimer-heterodimer interface in a manner similar to that in the activator-bound αγ heterodimer. This alteration not only stabilizes the active sites of both αQ139Aβ and αQ139Aγ heterodimers in active conformations but also induces conformational changes of the pseudo-allosteric site of the αQ139Aβ heterodimer enabling it to bind activators. In addition, the αQ139AICT+Ca+NADβNAD structure presents the first pseudo-Michaelis complex of HsIDH3, which allows us to identify the key residues involved in the binding of cofactor, substrate, and metal ion. Our structural and biochemical data together reveal new insights into the molecular mechanisms for allosteric regulation and the catalytic reaction of HsIDH3.

Information flow and allosteric communication in proteins.

Based on Schreiber's work on transfer entropy, a molecular theory of nonlinear information transfer between residue pairs in proteins is developed. The joint distribution function for residue fluctuations required by the theory is expressed in terms of tensor Hermite polynomials that conveniently separate harmonic and nonlinear contributions to information transfer. The harmonic part of information transfer is expressed as the difference between time dependent and independent mutual information. Third order nonlinearities are discussed in detail. The amount and speed of information transfer between residues, which are important for understanding allosteric activity in proteins, are discussed. Mutual information between two residues is commonly used for information transfer. While mutual information shows the maximum amount of information that may be transferred between two residues, it does not explain the actual amount of transfer nor the transfer rate of information. For this, dynamic equations of the system are needed. The solution of the Langevin equation and molecular dynamics trajectories are used in the present work for this purpose. Allosteric communication in human NAD-dependent isocitrate dehydrogenase is studied as an example. Calculations show that several paths contribute collectively to information transfer. Important residues on these paths are identified. Time resolved information transfer between these residues, their amplitudes, and transfer rates, which are in agreement with time resolved ultraviolet resonance Raman measurements in general, are estimated. Peak values of calculated information transfer, ∼0.01-0.04 bits, are about two orders of magnitude smaller than the information content of residues. They are comparable to mutual information values, however. Estimated transfer rates are in the order of 1-20 megabits per second, and sustained transfer during the activity time-span of proteins may be significant. Information transfer from third order contributions is one to two orders of magnitude smaller than the harmonic terms, showing that harmonic analysis is a good approximation to information transfer.

Structure and allosteric regulation of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase or HsIDH3 catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the TCA cycle. HsIDH3 exists and functions as a heterooctamer composed of the αβ and αγ heterodimers, and is regulated allosterically and/or competitively by numerous metabolites including CIT, ADP, ATP, and NADH. In this work, we report the crystal structure of HsIDH3 containing a β mutant in apo form. In the HsIDH3 structure, the αβ and αγ heterodimers form the α2βγ heterotetramer via their clasp domains, and two α2βγ heterotetramers form the (α2βγ)2 heterooctamer through insertion of the N-terminus of the γ subunit of one heterotetramer into the back cleft of the β subunit of the other heterotetramer. The functional roles of the key residues at the allosteric site, the pseudo allosteric site, the heterodimer and heterodimer–heterodimer interfaces, and the N-terminal of the γ subunit are validated by mutagenesis and kinetic studies. Our structural and biochemical data together demonstrate that the allosteric site plays an important role but the pseudo allosteric site plays no role in the allosteric activation of the enzyme; the activation signal from the allosteric site is transmitted to the active sites of both αβ and αγ heterodimers via the clasp domains; and the N-terminal of the γ subunit plays a critical role in the formation of the heterooctamer to ensure the optimal activity of the enzyme. These findings reveal the molecular mechanism of the assembly and allosteric regulation of HsIDH3.

Molecular mechanism of the dual regulatory roles of ATP on the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.

Molecular basis for the function of the αβ heterodimer of human NAD-dependent isocitrate dehydrogenase

Mammalian mitochondrial NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the tricarboxylic acid cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Different from the αγ heterodimer that can be allosterically activated by CIT and ADP, the αβ heterodimer cannot be allosterically regulated by the activators; however, the molecular mechanism is unclear. We report here the crystal structures of the αβ heterodimer of human NAD-IDH with the α subunit in apo form and in Ca2+-bound, NAD-bound, and NADH-bound forms. Structural analyses and comparisons reveal that the αβ heterodimer has a similar yet more compact overall structure compared with the αγ heterodimer and contains a pseudo-allosteric site that is structurally different from the allosteric site. In particular, the β3-α3 and β12-α8 loops of the β subunit at the pseudo-allosteric site adopt significantly different conformations from those of the γ subunit at the allosteric site and hence impede the binding of the activators, explaining why the αβ heterodimer cannot be allosterically regulated by the activators. The structural data also show that NADH can compete with NAD to bind to the active site and inhibits the activity of the αβ heterodimer. These findings together with the biochemical data reveal the molecular basis for the function of the αβ heterodimer of human NAD-IDH.

Insights into the inhibitory mechanisms of NADH on the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the α2βγ heterotetramer of NAD-IDH, the γ subunit plays the regulatory role and the β subunit the structural role. Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the αβ, αγ and α2βγ enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive αMgγ heterodimer. The NADH at the active site occupies the binding site for NAD+ and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD+ and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the αγ heterodimer by NADH.

The \u03b2 and \u03b3 subunits play distinct functional roles in the \u03b12\u03b2\u03b3 heterotetramer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase existing as the α2βγ heterotetramer, catalyzes the decarboxylation of isocitrate into α-ketoglutarate in the Krebs cycle, and is allosterically regulated by citrate, ADP and ATP. To explore the functional roles of the regulatory β and γ subunits, we systematically characterized the enzymatic properties of the holoenzyme and the composing αβ and αγ heterodimers in the absence and presence of regulators. The biochemical and mutagenesis data show that αβ and αγ alone have considerable basal activity but the full activity of α2βγ requires the assembly and cooperative function of both heterodimers. α2βγ and αγ can be activated by citrate or/and ADP, whereas αβ cannot. The binding of citrate or/and ADP decreases the S0.5,isocitrate and thus enhances the catalytic efficiencies of the enzymes, and the two activators can act independently or synergistically. Moreover, ATP can activate α2βγ and αγ at low concentration and inhibit the enzymes at high concentration, but has only inhibitory effect on αβ. Furthermore, the allosteric activation of α2βγ is through the γ subunit not the β subunit. These results demonstrate that the γ subunit plays regulatory role to activate the holoenzyme, and the β subunit the structural role to facilitate the assembly of the holoenzyme.

Molecular mechanism of the allosteric regulation of the \u03b1\u03b3 heterodimer of human NAD-dependent isocitrate dehydrogenase

Human NAD-dependent isocitrate dehydrogenase catalyzes the decarboxylation of isocitrate (ICT) into α-ketoglutarate in the Krebs cycle. It exists as the α2βγ heterotetramer composed of the αβ and αγ heterodimers. Previously, we have demonstrated biochemically that the α2βγ heterotetramer and αγ heterodimer can be allosterically activated by citrate (CIT) and ADP. In this work, we report the crystal structures of the αγ heterodimer with the γ subunit bound without or with different activators. Structural analyses show that CIT, ADP and Mg2+ bind adjacent to each other at the allosteric site. The CIT binding induces conformational changes at the allosteric site, which are transmitted to the active site through the heterodimer interface, leading to stabilization of the ICT binding at the active site and thus activation of the enzyme. The ADP binding induces no further conformational changes but enhances the CIT binding through Mg2+-mediated interactions, yielding a synergistic activation effect. ICT can also bind to the CIT-binding subsite, which induces similar conformational changes but exhibits a weaker activation effect. The functional roles of the key residues are verified by mutagenesis, kinetic and structural studies. Our structural and functional data together reveal the molecular mechanism of the allosteric regulation of the αγ heterodimer.

NAD-dependent isocitrate dehydrogenase as a novel target of tributyltin in human embryonic carcinoma cells.

Tributyltin (TBT) is known to cause developmental defects as endocrine disruptive chemicals (EDCs). At nanomoler concentrations, TBT actions were mediated by genomic pathways via PPAR/RXR. However, non-genomic target of TBT has not been elucidated. To investigate non-genomic TBT targets, we performed comprehensive metabolomic analyses using human embryonic carcinoma NT2/D1 cells. We found that 100 nM TBT reduced the amounts of α-ketoglutarate, succinate and malate. We further found that TBT decreased the activity of NAD-dependent isocitrate dehydrogenase (NAD-IDH), which catalyzes the conversion of isocitrate to α-ketoglutarate in the TCA cycle. In addition, TBT inhibited cell growth and enhanced neuronal differentiation through NAD-IDH inhibition. Furthermore, studies using bacterially expressed human NAD-IDH and in silico simulations suggest that TBT inhibits NAD-IDH due to a possible interaction. These results suggest that NAD-IDH is a novel non-genomic target of TBT at nanomolar levels. Thus, a metabolomic approach may provide new insights into the mechanism of EDC action.

Each Conserved Active Site Tyr in the Three Subunits of Human Isocitrate Dehydrogenase Has a Different Function

The human NAD-dependent isocitrate dehydrogenase (IDH) is a heterotetrameric mitochondrial enzyme with 2alpha:1beta:1gamma subunit ratio. The three subunits share 40-52% identity in amino acid sequence and each includes a tyrosine in a comparable position: alphaY126, betaY137, and gammaY135. To study the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated (one subunit at a time) to Ser, Phe, or Glu. Enzymes were expressed with one mutant and two wild-type subunits. The results of characterization of the mutant enzymes suggest that betaY137 is involved in NAD binding and allosteric activation by ADP. The alphaY126 is required for catalytic activity and likely acts as a general acid in the reaction. The gammaY135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions.

Role of α-Asp181, β-Asp192, and γ-Asp190 in the Distinctive Subunits of Human NAD-Specific Isocitrate Dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (IDH) is allosterically activated by ADP by lowering the Km for isocitrate. The enzyme has three subunit types with distinguishable sequences present in the approximate ratio 2alpha:1beta:1gamma and, per tetramer, binds 2 mol of each ligand. To evaluate whether the subunits also have distinct functions, we replaced equivalent aspartates, one subunit at a time, by asparagines; each expressed, purified enzyme was composed of one mutant and two wild-type subunits. The aspartates were chosen because beta-Asp192 and gamma-Asp190 had previously been affinity labeled by a reactive ADP analogue and alpha-Asp181 is equivalent based on sequence alignments. The alpha-D181N IDH mutant exhibits a 2000-fold decrease in Vmax, with increases of 15-fold in the Kms for Mn(II) and NAD and a much smaller change in the Km for isocitrate. In contrast, the Vmax values of the beta-D192N and gamma-D190N IDHs are only reduced 4-5-fold as compared to wild-type enzyme. The Km for NAD of the beta-D192N enzyme is 9 times that of the normal enzyme with little or no effect on the affinity for Mn(II) or isocitrate, while the Kms for coenzyme and for Mn(II) of the gamma-D190N enzyme are 19 and 72 times, respectively, that of the normal enzyme with a much smaller effect on the Km for isocitrate. Finally, all three mutant enzymes fail to respond to ADP by lowering the Km for isocitrate, although they do bind ADP. Thus, these aspartates are close to but not in the ADP site and are required for communication between the ADP and isocitrate sites. These results demonstrate that alpha-Asp181 is the only one of these aspartates essential for catalysis. Beta-Asp192 is a determinant of the enzyme's affinity for NAD, as is gamma-Asp190, while gamma-Asp190 also influences the enzyme's affinity for metal ion. We conclude that the NAD and ADP sites are shared between alpha- and beta- and alpha- and gamma-subunits, and the Mn(II) site is shared between alpha- and gamma-subunits, while the alpha-subunit is essential for catalysis. Although alpha-Asp181, beta-Asp192, and gamma-Asp190 may have derived from a common progenitor, these aspartates of the three subunits have evolved distinct functions.

Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.

Evaluation by Mutagenesis of the Importance of 3 Arginines in α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.