Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate.

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.     

The role of N286 and D320 in the reaction mechanism of human dihydrolipoamide dehydrogenase (E3) center domain

Summary According to the multiple alignment of various dihydrolipoamide dehydrogenases (E3s) sequences, three human mutant E3s of the conserved residues in the center domain, N286D, N286Q, and D320N were created, over-expressed and purified. We characterized these mutants to investigate the reaction mechanism of human dihydrolipoamide dehydrogenases. The specific activities of N286D, N286Q, and D320N are 30.84%, 24.57% and 48.60% to that of the wild-type E3 respectively. The FAD content analysis indicated that these mutant E3s about 96.0%, 99.4% and 82.7% of FAD content compared to that of wild-type E3 respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic’s data demonstrated that the K_cat of both forward and reverse reactions of these mutant proteins were decreased. These results suggest that N286 and D320 play a role in the catalytic function of the E3.     

Research note: Analysis of expressed sequence tags from the chrysophycean alga Ochromonas danica (Heterokontophyta)

A cDNA library was constructed from the chrysophycean alga, Ochromonas danica E. G. Pringsheim. 5′‐end sequencing of about 600 cDNA clones yielded 476 authentic expressed sequence tags (EST) of which 275 showed significant matches (E‐value <10^?4) to sequences in a public database. The annotation of these ESTs was carried out to assess subcellular localization of the putative proteins using several internet‐accessible prediction programs for subcellular localization. These analyses revealed that putative plastid proteins in Ochromonas possess N‐terminal bipartite presequences with a conserved phenylalanine at the N‐terminus of the predicted transit peptide‐like domains, similar to other ‘red‐lineage’ secondary symbiotic organisms. The examination of sequences of 3′‐UTR revealed that, similarly to chlorophyte algae, UGUAA may represent a putative polyadenylation signal in O. danica.     

Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E₂. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E₂ production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH₂, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.     

Domain structure and 1H-n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.     

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli.

I purified a new dihydrolipoamide dehydrogenase from a lpd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes. The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is different from the lpd gene product. The new dihydrolipoamide dehydrogenase had a molecular weight of 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was expressed in low amounts. It catalyzed the NAD+-dependent reduction of dihydrolipoamide with a maximal activity of 20 mumol/min per mg of protein and exhibited a hyperbolic dependence of catalytic activity on the concentration of both dihydrolipoamide and NAD+. The possible implication of the new dihydrolipoamide in the function of 2-oxo acid dehydrogenase complexes is discussed, as is its relation to binding protein-dependent transport.     

Catecholamines Enhance Dihydrolipoamide Dehydrogenase Inactivation by the Copper Fenton System. Enzyme Protection by Copper Chelators

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II) /H₂O₂ (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H₂O₂ and CA concentrations. Similar inhibitions were obtained wit! the assayed CAs and o-diphenols. CAs enhanced HO radical production by Cu(II) /H₂O₂, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipo amide, dihydrolipoic acid, DL-dithiothreitol, GSSG, try-panothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H₂O₂/CAs systems. Dena tured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH in creased and Captopril inhibited epinephrine oxidation by Cu(II)/H₂O₂ and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO^* from H₂O₂ by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the: enzyme active site by site-specifically generated HO^* radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.     

Oxidative stress‐induced structural changes in the microtubule‐associated flavoenzyme Irc15p from Saccharomyces cerevisiae

The genome of the yeast Saccharomyces cerevisiae encodes a canonical lipoamide dehydrogenase (Lpd1p) as part of the pyruvate dehydrogenase complex and a highly similar protein termed Irc15p (increased recombination centers 15). In contrast to Lpd1p, Irc15p lacks a pair of redox active cysteine residues required for the reduction of lipoamide and thus it is very unlikely that Irc15p performs a similar dithiol‐disulfide exchange reaction as reported for lipoamide dehydrogenases. We expressed IRC15 in Escherichia coli and purified the produced protein to conduct a detailed biochemical characterization. Here, we show that Irc15p is a dimeric protein with one FAD per protomer. Photoreduction of the protein generates the fully reduced hydroquinone without the occurrence of a flavin semiquinone radical. Similarly, reduction with NADH or NADPH yields the flavin hydroquinone without the occurrence of intermediates as observed for lipoamide dehydrogenase. The redox potential of Irc15p was ?313 ± 1 mV and is thus similar to lipoamide dehydrogenase. Reduced Irc15p is oxidized by several artificial electron acceptors such as potassium ferricyanide, 2,6‐dichlorophenol‐indophenol, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide, and menadione. However, disulfides such as cystine, glutathione, and lipoamide were unable to react with reduced Irc15p. Limited proteolysis and SAXS‐measurements revealed that the NADH‐dependent formation of hydrogen peroxide caused a substantial structural change in the dimeric protein. Therefore, we hypothesize that Irc15p undergoes a conformational change in the presence of elevated levels of hydrogen peroxide, which is a putative biomarker of oxidative stress. This conformational change may in turn modulate the interaction of Irc15p with other key players involved in regulating microtubule dynamics.     

The 8-isoprostaglandins: Evidence for eight compounds in human semen

Evidence is presented for the existence of a group of 8-iso prostaglandins in human semen, comprising 8-iso PG E₁^*, 8-iso PG E₂, 8-iso PG F_1α, 8-iso PG F_2α and the four corresponding 19-hydroxy prostaglandins. The E and F compounds have been positively identified by comparison of their mass spectra and chromatographic properties with those of authentic standards. Preliminary measurements of levels of these compounds in pooled semen are presented.     

Leishmania infantum: provision of reducing equivalents to the mitochondrial tryparedoxin/tryparedoxin peroxidase system

Within the mitochondrion of Leishmania infantum, hydroperoxide metabolism relies on the activity of tryparedoxin-dependent peroxidases (TXNPxs). Tryparedoxins (TXNs) are thioredoxin-related oxidoreductases, which in vitro are reduced by the trypanothione reductase/trypanothione [TR/T(SH)₂] redox couple. Still, there is no evidence that this actually occurs in the mitochondrion. This communication addresses the question of how the mitochondrial TXN/TXNPx system is reduced. First, using a digitonin fractionation assay, we show that TR activity is absent from the L. infantum mitochondrion. The possibility that this organelle possesses alternative electron sources for TXN/TXNPx is then investigated. Biochemical assays performed with purified recombinant enzymes, revealed that TR and T(SH)₂ can be replaced, albeit less efficiently, by the dihydrolipoamide dehydrogenase/lipoamide redox system as TXN/TXNPx electron donor. This result challenges the classical view that T(SH)₂ is the only reductant for TXNs and add new prospects regarding the involvement of 2-oxo acid dehydrogenase complexes in L. infantum mitochondrial hydroperoxide metabolism.     

Binding Interactions of Antagonists with 5‐Hydroxytryptamine3A Receptor Models

Abstract

Homology modeling was performed on the N‐terminal extracellular regions of human, mouse, and guinea pig 5‐hydroxytryptamine type 3A receptors (5‐HT₃R) based on the 24% sequence homology with and on the crystal structure of the snail acetylcholine binding protein (AChBP). Docking of 5‐HT₃ antagonists granisetron, tropisetron, ondansetron, dolasetron ('setrons), and (+)‐tubocurarine suggests an aromatic binding cleft behind a hydrophilic vestibule. Several intra‐ and interface interactions, H‐bonds, and salt bridges stabilize the pentameric structure and the binding cleft. The planar rings of antagonists are intercalated between aromatic side‐chains (W183‐Y234, Y143‐Y153). S227 donates H‐bonds to the carbonyl groups of 'setrons. The tertiary ammonium ions interact with E236, N128 or E129, and/or W90 (cation‐π interaction). This offers a molecular explanation of the pharmacophore models of 5‐HT₃R antagonists. Docking artifacts suggest some ambiguities in the binding loops A and C of the 5‐HT_3AR models. Lower potencies of (+)‐tubocurarine for human, and those of tropisetron for guinea pig 5‐HT_3ARs can be attributed to steric differences of I/S230 in the binding cleft and to distinct binding interactions with E229 and S227, respectively. Ligand binding interferes with crucial intra‐ and interface interactions along the binding cleft.     

Molecular modeling and functional confirmation of a predicted fatty acid binding site of mitochondrial aspartate aminotransferase

Molecular interactions are necessary for proteins to perform their functions. The identification of a putative plasma membrane fatty acid transporter as mitochondrial aspartate aminotransferase (mAsp-AT) indicated that the protein must have a fatty acid binding site. Molecular modeling suggests that such a site exists in the form of a 500-ų hydrophobic cleft on the surface of the molecule and identifies specific amino acid residues that are likely to be important for binding. The modeling and comparison with the cytosolic isoform indicated that two residues (Arg201 and Ala219) were likely to be important to the structure and function of the binding site. These residues were mutated to determine if they were essential to that function. Expression constructs with wild-type or mutated cDNAs were produced for bacteria and eukaryotic cells. Proteins expressed in Escherichia coli were tested for oleate binding affinity, which was decreased in the mutant proteins. 3T3 fibroblasts were transfected with expression constructs for both normal and mutated forms. Plasma membrane expression was documented by indirect immunofluorescence before [³H]oleic acid uptake kinetics were assayed. The V_max for uptake was significantly increased by overexpression of the wild-type protein but changed little after transfection with mutated proteins, despite their presence on the plasma membrane. The hydrophobic cleft in mAsp-AT can serve as a fatty acid binding site. Specific residues are essential for normal fatty acid binding, without which fatty acid uptake is compromised. These results confirm the function of this protein as a fatty acid binding protein.     

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Cloning and characterization of the Alcaligenes eutrophus 2-oxoglutarate dehydrogenase complex

Abstract Nucleotide sequence analysis of a 3.3-kb genomic Eco RI fragment and of relevant subfragments of a genomic 13.2-kb Sma I fragment of Alcaligenes eutrophus , which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative −35 / −10 promoter in the order odhA, odhB , and odhL . In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.     

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.     

A pH-Dependent Kinetic Model of Dihydrolipoamide Dehydrogenase from Multiple Organisms

Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD⁺ to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD⁺/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD⁺/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD⁺/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD⁺/NADH, and pH. These distributions were calculated using physiological NAD⁺/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.     

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.     

Syntheses of low-hemolytic antimicrobial gratisin peptides

Antibiotic and hemolytic activities of gratisin (GR), cyclo(-Val¹-Orn²-Leu³-d-Phe⁴-Pro⁵-d-Tyr⁶-)₂, and fifteen GR analogues, which have various d-amino acid residues in place of d-Tyr^6,6′ residues, were examined. Among them, [d-Orn^6,6′]-GR, [d-Lys^6,6′]-GR and [d-Arg^6,6′]-GR showed the strong activity against both Gram-positive and Gram-negative bacteria. In addition, the antibiotics showed significantly reduced toxicity against human blood cells compared with gramicidin S, cyclo(-Val¹-Orn²-Leu³-d-Phe⁴-Pro⁵-)₂.