Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica.

3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 m KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

Crystal structure of Haemophilus influenzae 3-isopropylmalate dehydrogenase (LeuB) in complex with the inhibitor O-isobutenyl oxalylhydroxamate.

3-isopropylmalate dehydrogenases (LeuB) belong to the leucine biosynthetic pathway and catalyze the irreversible oxidative decarboxylation of 3IPM to 2-ketoisocaproate that is finally converted into leucine by a branched-chain aminotransferase. Since leucine is an essential amino acid for humans, and it is also vital for the growth of many pathogenic bacteria, the enzymes belonging to this pathway can be considered as potential target sites for designing of a new class of antibacterial agents. We have determined the crystal structure of the Haemophilus influenzae LeuB in complex with the cofactor NAD+ and the inhibitor O-IbOHA, at 2.1 Å resolution; moreover, we have investigated the inhibitor mechanism of action by analyzing the enzyme kinetics. The structure of H. influenzae LeuB in complex with the intermediate analog inhibitor displays a fully closed conformation, resembling the previously observed, closed form of the equivalent enzyme of Thiobacillus ferrooxidans in complex with the 3IPM substrate. O-IbOHA was found to bind the active site by adopting the same conformation of 3IPM, and to induce an unreported repositioning of the side chain of the amino acids that participate in the coordination of the ligand. Indeed, the experimentally observed binding mode of O-IbOHA to the H. influenzae LeuB enzyme, reveals aspects of novelty compared to the computational binding prediction performed on M. tuberculosis LeuB. Overall, our data provide new insights for the structure-based rational design of a new class of antibiotics targeting the biosynthesis of leucine in pathogenic bacteria.

Similar structural stabilities of 3-isopropylmalate dehydrogenases from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 and its atmospheric congener S. oneidensis strain MR-1.

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ±â€¯0.1 MPa and a volume change of -225 ±â€¯24 mL mol-1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and -30 mL mol-1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: INSIGHTS ON LEUCINE AND ALIPHATIC GLUCOSINOLATE BIOSYNTHESIS.

Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different ß-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the ß-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.

Pressure adaptation of 3-isopropylmalate dehydrogenase from an extremely piezophilic bacterium is attributed to a single amino acid substitution.

3-Isopropylmalate dehydrogenase (IPMDH) from the extreme piezophile Shewanella benthica (SbIPMDH) is more pressure-tolerant than that from the atmospheric pressure-adapted Shewanella oneidensis (SoIPMDH). To understand the molecular mechanisms of this pressure tolerance, we analyzed mutated enzymes. The results indicate that only a single mutation at position 266, corresponding to Ala (SbIPMDH) and Ser (SoIPMDH), essentially affects activity under higher-pressure conditions. Structural analyses of SoIPMDH suggests that penetration of three water molecules into the cleft around Ser266 under high-pressure conditions could reduce the activity of the wild-type enzyme; however, no water molecule is observed in the Ala266 mutant.

Dual Role of the Active Site Residues of Thermus thermophilus 3-Isopropylmalate Dehydrogenase: Chemical Catalysis and Domain Closure.

The key active site residues K185, Y139, D217, D241, D245, and N102 of Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) have been replaced, one by one, with Ala. A drastic decrease in the kcat value (0.06% compared to that of the wild-type enzyme) has been observed for the K185A and D241A mutants. Similarly, the catalytic interactions (Km values) of these two mutants with the substrate IPM are weakened by more than 1 order of magnitude. The other mutants retained some (1-13%) of the catalytic activity of the wild-type enzyme and do not exhibit appreciable changes in the substrate Km values. The pH dependence of the wild-type enzyme activity (pK = 7.4) is shifted toward higher values for mutants K185A and D241A (pK values of 8.4 and 8.5, respectively). For the other mutants, smaller changes have been observed. Consequently, K185 and D241 may constitute a proton relay system that can assist in the abstraction of a proton from the OH group of IPM during catalysis. Molecular dynamics simulations provide strong support for the neutral character of K185 in the resting state of the enzyme, which implies that K185 abstracts the proton from the substrate and D241 assists the process via electrostatic interactions with K185. Quantum mechanics/molecular mechanics calculations revealed a significant increase in the activation energy of the hydride transfer of the redox step for both D217A and D241A mutants. Crystal structure analysis of the molecular contacts of the investigated residues in the enzyme-substrate complex revealed their additional importance (in particular that of K185, D217, and D241) in stabilizing the domain-closed active conformation. In accordance with this, small-angle X-ray scattering measurements indicated the complete absence of domain closure in the cases of D217A and D241A mutants, while only partial domain closure could be detected for the other mutants. This suggests that the same residues that are important for catalysis are also essential for inducing domain closure.

Deciphering the Dynamics of Non-Covalent Interactions Affecting Thermal Stability of a Protein: Molecular Dynamics Study on Point Mutant of Thermus thermophilus Isopropylmalate Dehydrogenase.

Thermus thermophilius isopropylmalate dehydrogenase catalyzes oxidative decarboxylation and dehydrogenation of isopropylmalate. Substitution of leucine to alanine at position 172 enhances the thermal stability among the known point mutants. Exploring the dynamic properties of non-covalent interactions such as saltbridges, hydrogen bonds and hydrophobic interactions to explain thermal stability of a protein is interesting in its own right. In this study dynamic changes in the non-covalent interactions are studied to decipher the deterministic features of thermal stability of a protein considering a case study of a point mutant in Thermus thermophilus isopropylmalate dehydrogenase. A total of four molecular dynamic simulations of 0.2 µs were carried out on wild type and mutant's functional dimers at 300 K and 337 K. Higher thermal stability of the mutant as compared to wild type is revealed by root mean square deviation, root mean square fluctuations and Cα-Cα distance with an increase in temperature from 300 K to 337 K. Most of the regions of wild type fluctuate higher than the corresponding regions of mutant with an increase in temperature. Cα-Cα distance analysis suggests that long distance networks are significantly affected in wild type as compared to the mutant. Short lived contacts are higher in wild type, while long lived contacts are lost at 337 K. The mutant forms less hydrogen bonds with water as compared to wild type at 337 K. In contrast to wild type, the mutant shows significant increase in unique saltbridges, hydrogen bonds and hydrophobic contacts at 337 K. The current study indicates that there is a strong inter-dependence of thermal stability on the way in which non-covalent interactions reorganize, and it is rewarding to explore this connection in single mutant studies.

Glutamate 270 plays an essential role in K(+)-activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase.

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K(+) compared to the wild-type enzyme. A 3-4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K(+). SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K(+) into close proximity of the nicotinamide ring of NAD(+) and the electron-withdrawing effect of K(+) may help to polarise the aromatic ring in order to aid the hydride-transfer.

Structural and energetic basis of isopropylmalate dehydrogenase enzyme catalysis.

UNLABELLED: The three-dimensional structure of the enzyme 3-isopropylmalate dehydrogenase from the bacterium Thermus thermophilus in complex with Mn(2+) , its substrate isopropylmalate and its co-factor product NADH at 2.0 Å resolution features a fully closed conformation of the enzyme. Upon closure of the two domains, the substrate and the co-factor are brought into precise relative orientation and close proximity, with a distance between the C2 atom of the substrate and the C4N atom of the pyridine ring of the co-factor of approximately 3.0 Å. The structure further shows binding of a K(+) ion close to the active site, and provides an explanation for its known activating effect. Hence, this structure is an excellent mimic for the enzymatically competent complex. Using high-level QM/MM calculations, it may be demonstrated that, in the observed arrangement of the reactants, transfer of a hydride from the C2 atom of 3-isopropylmalate to the C4N atom of the pyridine ring of NAD(+) is easily possible, with an activation energy of approximately 15 kcal·mol(-1) . The activation energy increases by approximately 4-6 kcal·mol(-1) when the K(+) ion is omitted from the calculations. In the most plausible scenario, prior to hydride transfer the ε-amino group of Lys185 acts as a general base in the reaction, aiding the deprotonation reaction of 3-isopropylmalate prior to hydride transfer by employing a low-barrier proton shuttle mechanism involving a water molecule. DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4F7I.

Pressure effects on the chimeric 3-isopropylmalate dehydrogenases of the deep-sea piezophilic Shewanella benthica and the atmospheric pressure-adapted Shewanella oneidensis.

The chimeric 3-isopropylmalate dehydrogenase enzymes were constructed from the deep-sea piezophilic Shewanella benthica and the shallow water Shewanella oneidensis genes. The properties of the enzymatic activities under pressure conditions indicated that the central region, which contained the active center and the dimer forming domains, was shown to be the most important region for pressure tolerance in the deep-sea enzyme.

Drugs against Mycobacterium tuberculosis 3-isopropylmalate dehydrogenase can be developed using homologous enzymes as surrogate targets.

3-Isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (Mtb) may be a target for specific drugs against this pathogenic bacterium. We have expressed and purified Mtb IPMDH and determined its physicalchemical and enzymological properties. Size-exclusion chromatography and dynamic light scattering measurements (DLS) suggest a tetrameric structure for Mtb IPMDH, in contrast to the dimeric structure of most IPMDHs. The kinetic properties (kcat and Km values) of Mtb IPMDH and the pH-dependence of kcat are very similar to both Escherichia coli (Ec) and Thermus thermophilus (Tt) IPMDHs. The stability of Mtb IPMDH in 8 M urea is close to that of the mesophilic counterpart, Ec IPMDH, both of them being much less stable than the thermophilic (Tt) enzyme. Two known IPMDH inhibitors, O-methyl oxalohydroxamate and 3-methylmercaptomalate, have been synthesised. Their inhibitory effects were found to be independent of the origin of IPMDHs. Thus, experiments with either Ec or Tt IPMDH would be equally relevant for designing specific inhibitory drugs against Mtb IPMDH.

High-throughput analysis of in vivo protein stability.

Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with high-throughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein. The variants are fused to a metabolic enzyme, which here is the yeast Leu2 protein. Plasmids encoding these Leu2 fusion proteins are transformed into yeast, with the resultant fusion proteins accumulating to different levels based on their stability and leading to different doubling times when the yeast are grown in the absence of leucine. Sequencing of an input population of variants of a protein and the population of variants after leucine selection allows the stability of tens of thousands of variants to be scored in parallel. By applying the Stable-seq method to variants of the protein degradation signal Deg1 from the yeast Matα2 protein, we generated a high-resolution map that reveals the effect of ∼30,000 mutations on protein stability. We identified mutations that likely affect stability by changing the activity of the degron, by leading to translation from new start codons, or by affecting N-terminal processing. Stable-seq should be applicable to other organisms via the use of suitable reporter proteins, as well as to the analysis of complex mixtures of fusion proteins.

Transient kinetic studies reveal isomerization steps along the kinetic pathway of Thermus thermophilus 3-isopropylmalate dehydrogenase.

To identify the rate-limiting step(s) of the 3-isopropylmalate dehydrogenase-catalysed reaction, time courses of NADH production were followed by stopped flow (SF) and quenched flow (QF). The steady state kcat and Km values did not vary between enzyme concentrations of 0.1 and 20 µm. A burst phase of NADH formation was shown by QF, indicating that the rate-limiting step occurs after the redox step. The kinetics of protein conformational change(s) induced by the complex of 3-isopropylmalate with Mg(2+) were followed by using the fluorescence resonance energy transfer signal between protein tryptophan(s) and the bound NADH. A reaction scheme was proposed by incorporating the rate constant of a fast protein conformational change (possibly domain closure) derived from the separately recorded time-dependent formation of the fluorescence resonance energy transfer signal. The rate-limiting step seems to be another slower conformational change (domain opening) that allows product release.

Structural analysis of 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 and the nonpiezophile Shewanella oneidensis MR-1.

Organisms living in deep seas such as the Mariana Trench must be adapted to the extremely high pressure environment. For example, the 3-isopropylmalate dehydrogenase from the obligate piezophile Shewanella benthica DB21MT-2 (SbIPMDH) remains active in extreme conditions under which that from the land bacterium S. oneidensis MR-1 (SoIPMDH) becomes inactivated. In order to unravel the differences between these two IPMDHs, their structures were determined at ~1.5 Å resolution. Comparison of the structures of the two enzymes shows that SbIPMDH is in a more open form and has a larger internal cavity volume than SoIPMDH at atmospheric pressure. This loosely packed structure of SbIPMDH could help it to avoid pressure-induced distortion of the native structure and to remain active at higher pressures than SoIPMDH.

High-pressure-induced water penetration into 3-isopropylmalate dehydrogenase.

Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Šresolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

Substitutions of coenzyme-binding, nonpolar residues improve the low-temperature activity of thermophilic dehydrogenases.

Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD(+), suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.

Essential role of the metal-ion in the IPM-assisted domain closure of 3-isopropylmalate dehydrogenase.

X-ray structures of 3-isopropylmalate dehydrogenase (IPMDH) do not provide sufficient information on the role of the metal-ion in the metal-IPM assisted domain closure. Here solution studies were carried out to test its importance. Small-angle X-ray scattering (SAXS) experiments with the Thermus thermophilus enzyme (complexes with single substrates) have revealed only a very marginal (0-5%) extent of domain closure in the absence of the metal-ion. Only the metal-IPM complex, but neither the metal-ion nor the free IPM itself, is efficient in stabilizing the native protein conformation as confirmed by denaturation experiments with Escherichia coli IPMDH and by studies of the characteristic fluorescence resonance energy transfer (FRET) signal (from Trp to bound NADH) with both IPMDHs. A possible atomic level explanation of the metal-effect is given.

Atomic level description of the domain closure in a dimeric enzyme: thermus thermophilus 3-isopropylmalate dehydrogenase.

The domain closure associated with the catalytic cycle is described at an atomic level, based on pairwise comparison of the X-ray structures of homodimeric Thermus thermophilus isopropylmalate dehydrogenase (IPMDH), and on their detailed molecular graphical analysis. The structures of the apo-form without substrate and in complex with the divalent metal-ion to 1.8 Å resolution, in complexes with both Mn(2+) and 3-isopropylmalate (IPM), as well as with both Mn(2+) and NADH, were determined at resolutions ranging from 2.0 to 2.5 Å. Single crystal microspectrophotometric measurements demonstrated the presence of a functionally competent protein conformation in the crystal grown in the presence of Mn(2+) and IPM. Structural comparison of the various complexes clearly revealed the relative movement of the two domains within each subunit and allowed the identification of two hinges at the interdomain region: hinge 1 between αd and ßF as well as hinge 2 between αh and ßE. A detailed analysis of the atomic contacts of the conserved amino acid side-chains suggests a possible operational mechanism of these molecular hinges upon the action of the substrates. The interactions of the protein with Mn(2+) and IPM are mainly responsible for the domain closure: upon binding into the cleft of the interdomain region, the substrate IPM induces a relative movement of the secondary structural elements ßE, ßF, ßG, αd and αh. A further special feature of the conformational change is the movement of the loop bearing the amino acid Tyr139 that precedes the interacting arm of the subunit. The tyrosyl ring rotates and moves by at least 5 Å upon IPM-binding. Thereby, new hydrophobic interactions are formed above the buried isopropyl-group of IPM. Domain closure is then completed only through subunit interactions: a loop of one subunit that is inserted into the interdomain cavity of the other subunit extends the area with the hydrophobic interactions, providing an example of the cooperativity between interdomain and intersubunit interactions.

Enhancement of the latent 3-isopropylmalate dehydrogenase activity of promiscuous homoisocitrate dehydrogenase by directed evolution.

HICDH (homoisocitrate dehydrogenase), which is involved in lysine biosynthesis through α-aminoadipate, is a paralogue of IPMDH [3-IPM (3-isopropylmalate) dehydrogenase], which is involved in leucine biosynthesis. TtHICDH (Thermus thermophilus HICDH) can recognize isocitrate, as well as homoisocitrate, as the substrate, and also shows IPMDH activity, although at a considerably decreased rate. In the present study, the promiscuous TtHICDH was evolved into an enzyme showing distinct IPMDH activity by directed evolution using a DNA-shuffling technique. Through five repeats of DNA shuffling/screening, variants that allowed Escherichia coli C600 (leuB⁻) to grow on a minimal medium in 2 days were obtained. One of the variants LR5-1, with eight amino acid replacements, was found to possess a 65-fold increased k(cat)/K(m) value for 3-IPM, compared with TtHICDH. Introduction of a single back-replacement H15Y change caused a further increase in the k(cat)/K(m) value and a partial recovery of the decreased thermotolerance of LR5-1. Site-directed mutagenesis revealed that most of the amino acid replacements found in LR5-1 effectively increased IPMDH activity; replacements around the substrate-binding site contributed to the improved recognition for 3-IPM, and other replacements at sites away from the substrate-binding site enhanced the turnover number for the IPMDH reaction. The crystal structure of LR5-1 was determined at 2.4 Å resolution and revealed that helix α4 was displaced in a manner suitable for recognition of the hydrophobic γ-moiety of 3-IPM. On the basis of the crystal structure, possible reasons for enhancement of the turnover number are discussed.

Crystallization and preliminary X-ray diffraction analysis of various enzyme-substrate complexes of isopropylmalate dehydrogenase from Thermus thermophilus.

The Thermus thermophilus 3-isopropylmalate dehydrogenase (Tt-IPMDH) enzyme catalyses the penultimate step of the leucine-biosynthesis pathway. It converts (2R,3S)-3-isopropylmalate to (2S)-2-isopropyl-3-oxosuccinate in the presence of divalent Mg(2+) or Mn(2+) and with the help of NAD(+). In order to elucidate the detailed structural and functional mode of the enzymatic reaction, crystals of Tt-IPMDH were grown in the presence of various combinations of substrate and/or cofactors. Here, the crystallization, data collection and preliminary crystallographic analyses of six such complexes are reported.