Crystal structure of human dihydrolipoamide dehydrogenase: NAD+/NADH binding and the structural basis of disease-causing mutations

Human dihydrolipoamide dehydrogenase (hE3) is an enzymatic component common to the mitochondrial alpha-ketoacid dehydrogenase and glycine decarboxylase complexes. Mutations to this homodimeric flavoprotein cause the often-fatal human disease known as E3 deficiency. To catalyze the oxidation of dihydrolipoamide, hE3 uses two molecules: non-covalently bound FAD and a transiently bound substrate, NAD+. To address the catalytic mechanism of hE3 and the structural basis for E3 deficiency, the crystal structures of hE3 in the presence of NAD+ or NADH have been determined at resolutions of 2.5A and 2.1A, respectively. Although the overall fold of the enzyme is similar to that of yeast E3, these two structures differ at two loops that protrude from the proteins and at their FAD-binding sites. The structure of oxidized hE3 with NAD+ bound demonstrates that the nicotinamide moiety is not proximal to the FAD. When NADH is present, however, the nicotinamide base stacks directly on the isoalloxazine ring system of the FAD. This is the first time that this mechanistically requisite conformation of NAD+ or NADH has been observed in E3 from any species. Because E3 structures were previously available only from unicellular organisms, speculations regarding the molecular mechanisms of E3 deficiency were based on homology models. The current hE3 structures show directly that the disease-causing mutations occur at three locations in the human enzyme: the dimer interface, the active site, and the FAD and NAD(+)-binding sites. The mechanisms by which these mutations impede the function of hE3 are discussed.     

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.     

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.     

Exercise-induced activation of the branched-chain 2-oxo acid dehydrogenase in human muscle

The present study was conducted to investigate the metabolic regulation of the oxidation of branched-chain amino acids (BCAA) by exercise in human skeletal muscle. Five trained male volunteers were exercised on a cycle ergometer at 70% +/- 10% (mean +/- SD) of their maximal oxygen consumption (VO2max). Percutaneous quadriceps muscle biopsies were obtained under local anaesthesia at rest and after 30 and 120 min of exercise. In the muscle samples the active and total amount of the branched-chain 2-oxo acid dehydrogenase complex (BC-complex), the regulatory enzyme in the oxidative pathway of the BCAA, were measured. Glycogen content and activity of mitochondrial marker enzymes were also measured. Blood samples were obtained every 20 min for the measurement of metabolites. Heart rate and rated perceived exertion on the Borg scale were recorded every 10 min. At rest 4.0% +/- 2.5% of the BC complex was active, after 30 min of exercise 9.9% +/- 9.0% and after 120 min 17.5% +/- 8.5% (mean +/- SD). Exercise did not change the total activity. The largest activation was seen in two of the subjects who developed higher blood lactates early on during exercise and decreased their muscle glycogen more (indications of anaerobic metabolism). These data demonstrate that in trained individuals significant increases in the activity of the BC-complex occur only after prolonged intense exercise. In spite of the 4-fold activation, the data support the classical view that amino acids and protein do not contribute substantially as an energy source during exercise, since VO2 increased more than 20-fold.     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.     

Roles of cysteine residues in the inhibition of human glutamate dehydrogenase by palmitoyl-CoA

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the cassette mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl-CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA. [BMB Reports 2012; 45(12): 707-712]     

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase

Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate- and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations.     

Computational study of conformational changes in human 3-hydroxy-3-methylglutaryl coenzyme reductase induced by substrate binding

Abstract

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is mainly involved in the regulation of cholesterol biosynthesis. HMGR catalyses the reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonate at the expense of two NADPH molecules in a two-step reversible reaction. In the present study, we constructed a model of human HMGR (hHMGR) to explore the conformational changes of HMGR in complex with HMG-CoA and NADPH. In addition, we analysed the complete sequence of the Flap domain using molecular dynamics (MD) simulations and principal component analysis (PCA). The simulations revealed that the Flap domain plays an important role in catalytic site activation and substrate binding. The apo form of hHMGR remained in an open state, while a substrate-induced closure of the Flap domain was observed for holo hHMGR. Our study also demonstrated that the phosphorylation of Ser872 induces significant conformational changes in the Flap domain that lead to a complete closure of the active site, suggesting three principal conformations for the first stage of hHMGR catalysis. Our results were consistent with previous proposed models for the catalytic mechanism of hHMGR.

Communicated by Ramaswamy H. Sarma     

15-Hydroxyprostaglandin dehydrogenase can be induced by dexamethasone and other glucocorticoids at the therapeutic level in A549 human lung adenocarcinoma cells

Dexamethasone induced the expression of 15-PGDH in a time- and concentration-dependent manner in A549 human lung adenocarcinoma cells. Maximal induction was observed at 10nM. Induction of 15-PGDH expression was also achieved by other synthetic glucocorticoids. Induction was inhibited by the addition of pro-inflammatory cytokines and phorbol ester. These pro-inflammatory agents were also shown to induce COX-2 expression. PMA was found to be the most effective stimulator of COX-2 expression and the most potent inhibitor of dexamethasone-induced 15-PGDH expression. Attenuation of dexamethasone-induced 15-PGDH expression by PMA was, in part, due to a protein kinase C-mediated mechanism. The induction of 15-PGDH expression by dexamethasone was blocked by a glucocorticoid receptor antagonist RU 486 and by a nuclear translocation inhibitor geldanamycin, indicating that the induction is a genetic mechanism. The induction of 15-PGDH expression by dexamethasone and other glucocorticoids at the therapeutic level provides an additional biochemical mechanism for the anti-inflammatory action of these glucocorticoids.     

An NMR study of O-glycosylation induced structural changes in the alpha-helix of calcitonin

We previously reported that two out of seven artificially O-glycosylated calcitonin derivatives had an altered peptide backbone conformation as indicated by decreased helical contents, determined by CD measurement. In the present study, two of those derivatives, in which a GalNAc residue is attached to Thr6 or Thr21 of calcitonin, were analyzed by NMR in order to determine the structural changes induced by the O-glycosylation in more detail. Deviations in the chemical shifts suggest that the structural change is not global but only a local one and is located in the vicinity of each O-glycosylation site. The intensities of the NOE cross peaks, an indicator of -helical structure, also were decreased around the O-glycosylation site. The hydrogen/deuterium exchange rates of the main chain amide protons increased at the N- or C-terminal portion of the -helix corresponding to the respective O-glycosylation site and explains the results of the CD experiments. The inter-residual NOE cross peaks between the carbohydrate and the peptide portions, other than the O-glycosylated amino acid residue, showed that local structural contacts extended three or two residue distance for Thr6- or Thr21-glycosylated derivative, respectively. Thus, we conclude that the O-glycosylation induced a change in the local structure and that this structural perturbation modulated the original -helical structure of calcitonin, resulting in the apparent decrease in the helical content deduced from CD spectra.     

Novel allosteric properties produced by residue substitutions in the subunit interface of yeast NAD+-specific isocitrate dehydrogenase

Yeast NAD+-specific isocitrate dehydrogenase (IDH) is an octamer of four IDH1 and four IDH2 subunits, and the basic structural unit of the enzyme is an IDH1/IDH2 heterodimer. To investigate one aspect of the interaction between IDH1 and IDH2, residues in a hydrophobic region at the heterodimer interface (Val-216, Ser-220, and Val-224 in IDH1; Ile-221, Val-225, and Val-229 in IDH2) were replaced by alanine residues in each and in both subunits. Gel filtration and sedimentation velocity analyses demonstrated that the residue substitutions do not disrupt the octameric structure of IDH. However, these substitutions produce novel kinetic properties including, with respect to cofactor, positive allosteric regulation by AMP and cooperativity in the absence of AMP. These allosteric properties are also apparent in NAD+-binding experiments. Despite substantial measurable activity for the mutant enzyme containing residue substitutions in both subunits, expression of this enzyme produces growth phenotypes indicative of IDH dysfunction in vivo.     

Single point mutation induced alterations in the equilibrium structural transitions on the folding landscape of HIV-1 protease

Equilibrium folding–unfolding transitions are hard to study in HIV-1 protease (PR) because of its autolytic properties. Further, the protease exhibits many tolerant point mutations some of which also impart drug resistance to the protein. It is conceivable that the mutations affect protein's function by altering its folding characteristics; these would clearly depend on the nature of the mutations themselves. In this background, we report here NMR studies on the effects of D25?N mutation, which removes one negative charge from the protein at the active site, on the equilibrium folding behaviour of PR starting from its acetic acid denatured state. It is observed that in PR_D25N two slowly exchanging conformations are present at the N-terminal. One of them is similar to that of PR. Though the conformational and dynamics preferences of PR and PR_D25N are fairly similar in 9?M acetic acid, they seem to undergo different folding transitions when acetic acid concentration is reduced. The differences are seen in the active site, in the flap, and in the hinge of the flap regions. The present study suggests that such differences, though different in detail, would occur for other mutations as well, and also for different initial denatured states. These would have significant regulatory implications for the efficacy of protease function.     

A genetic and molecular analysis of P-induced mutations at the glucose-6-phosphate dehydrogenase locus in Drosophila melanogaster

Summary We examined P factor induced mutations of the Zw gene of Drosophila melanogaster in order to learn more about the site specificity of such mutations. Approximately 70000 chromosomes were screened using a powerful positive selection scheme. As only two mutants were discovered, Zw is a cold spot for transposable element insertion. One mutation involved a complex P element associated chromosomal rearrangement which was used to define the orientation of the gene with respect to the centromere of the X chromosome. The second mutation was either a simple, non-dysgenically induced point mutation or a very unstable insertion.     

Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis

A new mediated amperometric biosensor for fructose is described. The sensor is based on a commercially available D-fructose dehydrogenase. The enzyme is incorporated in a carbon paste matrix containing Os(bpy)₂Cl₂ as redox mediator that achieves electron transfer at 0·1 V (versus Ag/AgCl) with maximum apparent current densities of 1·2 mA/cm². The dependence of the steady-state current on the loading of the mediator and the enzyme, other electrode construction parameters, the operating potential, the pH and the temperature was studied. In the steady-state mode the response current was directly proportional to D-fructose concentration from 0·2 to 20mM with a detection limit of 35 μM (signal-to-noise ratio, S/N, 3). In the flow injection analysis mode the response current was directly proportional to D-fructose concentration from 0·5 to 15 M with a detection limit of 115 μM (S/N 3). The sensor was used for the determination of fructose in food samples in a flow injection system and validated with a commercial enzyme kit.     

Fractal dimension analysis and mathematical morphology of structural changes in actin filaments imaged by electron microscopy

In this work, we examined structural changes of actin filaments interacting with myosin visualized by quick freeze deep-etch replica electron microscopy (EM) by using a new method of image processing/analysis based on mathematical morphology.In order to quantify the degree of structural changes, two characteristic patterns were extracted from the EM images. One is the winding pattern of the filament shape (WP) reflecting flexibility of the filament, and the other is the surface pattern of the filament (SP) reflecting intra-molecular domain-mobility of actin monomers constituting the filament. EM images were processed by morphological filtering followed by box-counting to calculate the fractal dimensions for WP (D_WP) and SP (D_SP). The result indicates that D_WP was larger than D_SP irrespective of the state of the filament (myosin-free or bound) and that both parameters for myosin-bound filaments were significantly larger than those for myosin-free filaments. Overall, this work provides the first quantitative insight into how conformational disorder of actin monomers is correlated with the myosin-induced increase in flexibility of actin filaments along their length as suggested by earlier studies with different techniques. Our method is yet to be improved in details, but promising as a powerful tool for studying the structural change of protein molecules and their assemblies, which can potentially be applied to a wide range of biological and biomedical images.     

Spectral broadening of the Soret band in myoglobin: an interpretation by the full spectrum of low-frequency modes from a normal modes analysis

In this work the temperature dependence of the Soret band line shape in carbon-monoxy myoglobin is re-analyzed by using both the full correlator approach in the time domain and the frequency domain approach. The new analyses exploit the full density of vibrational states of carbon-monoxy myoglobin available from normal modes analysis, and avoid the artificial division of the entire set of vibrational modes coupled to the Soret transition into "high-frequency" and "low-frequency" subsets; the frequency domain analysis, however, makes use of the so-called short-times approximation, while the time domain one avoids it. Time domain and frequency domain analyses give very similar results, thus supporting the applicability of the short-times approximation to the analysis of hemeprotein spectra; in particular, they clearly indicate the presence of spectral heterogeneity in the Soret band of carbon-monoxy myoglobin. The analyses also show that a temperature dependence of the Gaussian width parameter steeper than the hyperbolic cotangent law predicted by the Einstein harmonic oscillator and/or a temperature dependence of inhomogeneous broadening are not sufficient to obtain quantitative information on the magnitude of an-harmonic contributions to the iron-heme plane motion. However, the dependence of the previous two quantities may be used to obtain semiquantitative information on the overall coupling of the Soret transition to the low-frequency modes and therefore on the dynamic properties of the heme pocket in different states of the protein.     

Specific damage induced by X-ray radiation and structural changes in the primary photoreaction of bacteriorhodopsin

Bacteriorhodopsin, the sole membrane protein of the purple membrane of Halobacterium salinarum, functions as a light-driven proton pump. A 3-D crystal of bacteriorhodopsin, which was prepared by the membrane fusion method, was used to investigate structural changes in the primary photoreaction. It was observed that when a frozen crystal was exposed to a low flux of X-ray radiation (5 x 10(14)photons mm(-2)), nearly half of the protein was converted into an orange species, exhibiting absorption peaks at 450 nm, 478 nm and 510 nm. The remainder retained the normal photochemical activity until Asp85 in the active site was decarboxlyated by a higher flux of X-ray radiation (10(16)photons mm(-2)). The procedure of diffraction measurement was improved so as to minimize the effects of the radiation damage and determine the true structural change associated with the primary photoreaction. Our structural model of the K intermediate indicates that the Schiff base linkage and the adjacent bonds in the polyene chain of retinal are largely twisted so that the Schiff base nitrogen atom still interacts with a water molecule located near Asp85. With respect to the other part of the protein, no appreciable displacement is induced in the primary photoreaction.