Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9. 5%. The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit. The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm. Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH. Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase. The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier. The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism. The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion. The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form. The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences. The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes. The gene was named lpd. All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.
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PMID:Lipoamide dehydrogenase from streptomyces seoulensis: biochemical and genetic properties. 985 75

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.
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PMID:Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate. 1033 57

Enzymes from extreme halophiles have potential as catalysts in biotransformations. We have developed methods for the expression in Escherichia coli and purification of two enzymes from Haloferax volcanii: dihydrolipoamide dehydrogenase and citrate synthase. Both enzymes were expressed in E. coli using the cytoplasmic expression vectors, pET3a and pET3d. Citrate synthase was soluble and inactive, whereas dihydrolipoamide dehydrogenase was expressed as inclusion bodies. Citrate synthase was reactivated following overnight incubation in 2 M KCl, and dihydrolipoamide dehydrogenase was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing 2 M KCl, 10 microM FAD, 1 mM NAD, and 0.3 mM GSSG/3 mM GSH. Maximal activity was obtained after 3 days incubation at 4 degrees C. Purification of the two active enzymes was carried out using high-resolution methods. Dihydrolipoamide dehydrogenase was purified using copper-based metal ion affinity chromatography in the presence of 2 M KCl. Citrate synthase was recovered using dye-affinity chromatography in the presence of salt. A high yield of active enzyme was obtained in both cases. Following purification, characterisation of both recombinant proteins showed that their kinetics and salt-dependence were comparable to those of the native enzymes. Expression of active protein was attempted both by growth of E. coli in the presence of salt and betaine, and also by using periplasmic expression vectors in combination with a high salt growth media. Neither strategy was successful.
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PMID:Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli. 1039 37

Ferric leghemoglobin reductase (FLbR), an enzyme reducing ferric leghemoglobin (Lb) to ferrous Lb, was purified from cowpea (Vigna unguiculata) root nodules by sequential chromatography on hydroxylapatite followed by Mono-Q HR5/5 FPLC and Sephacryl S-200 gel filtration. The purified cowpea FLbR had a specific activity of 216 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for cowpea Lb(3+) and a specific activity of 184 nmol Lb(2+)O(2) formed min(-1) mg(-1) of enzyme for soybean Lb(3+). A cDNA clone of cowpea FLbR was obtained by screening a cowpea root nodule cDNA library. The nucleotide sequence of cowpea FLbR cDNA exhibited about 88% similarity with soybean (Glycine max) FLbR and 85% with pea (Pisum sativum) dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) cDNAs. Conserved regions for the FAD-binding site, NAD(P)H-binding site, and disulfide active site were identified among the deduced amino acid sequences of cowpea FLbR, soybean FLbR, pea DLDH and other enzymes in the family of the pyridine nucleotide-disulfide oxido-reductases.
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PMID:Analysis of a ferric leghemoglobin reductase from cowpea (Vigna unguiculata) root nodules. 1072 15

Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 m guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization. This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65 degrees C for 15 min. The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD. Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization. Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway.
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PMID:FAD insertion is essential for attaining the assembly competence of the dihydrolipoamide dehydrogenase (E3) monomer from Escherichia coli. 1097 Aug 89

Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c). To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c. Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79 degrees C and 90 degrees C, respectively. Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate. E3d lost FAD, and inactive E3d polypeptides were aggregated. The sulfonate bound to the aggregate became notably fluorescent. The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex. Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.
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PMID:Thermally induced disintegration of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase. 1105 97

Lipoamide dehydrogenase belongs to a family of pyridine nucleotide disulfide oxidoreductases and is ubiquitous in aerobic organisms. This enzyme also reduces ubiquinone (the only endogenously synthesized lipid-soluble antioxidant) to ubiquinol, the form in which it functions as an antioxidant. The reduction of ubiquinone was linear with time and exhibited turnover numbers of 5 and 1.2 min(-1) in the presence and absence of zinc, respectively. The reaction was stimulated by zinc and cadmium but not by the other divalent ions tested. The zinc/cadmium-dependent stimulation of the reaction increased rapidly and linearly up to a concentration of 0.1 mM and was even further increased at 0.5 mM. At pH 6, the activity was three times higher than at physiological pH. Alteration of the NADPH : NADP(+) ratio revealed that the reaction is inhibited by higher concentrations of the oxidized cofactors. FAD reduced ubiquinone in a dose-dependent manner at a considerably lower rate, suggesting that the reduction of ubiquinone by lipoamide dehydrogenase involves the FAD moiety of the enzyme.
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PMID:Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway. 1123 2

The gene encoding dihydrolipoamide dehydrogenase from Mycobacterium tuberculosis, Rv0462, was expressed in Escherichia coli and the protein purified to homogeneity. The 49 kDa polypeptide forms a homodimer containing one tightly bound molecule of FAD/monomer. The results of steady-state kinetic analyses using several reduced pyridine nucleotide analogs and a variety of electron acceptors, and the ability of the enzyme to catalyze the transhydrogenation of NADH and thio-NAD(+) in the absence of D,L-lipoamide, demonstrated that the enzyme uses a ping-pong kinetic mechanism. Primary deuterium kinetic isotope effects on V and V/K at pH 7.5 using NADH deuterated at the C(4)-proS position of the nicotinamide ring are small [(D)(V/K)(NADH) = 1.12 +/- 0.15, (D)V(app) = 1.05 +/- 0.07] when D,L-lipoamide is the oxidant but large and equivalent [(D)(V/K)(NADH) = (D)V = 2.95 +/- 0.03] when 5-hydroxy-1,4-naphthoquinone is the oxidant. Solvent deuterium kinetic isotope effects at pH 5.8, using APADH as the reductant, are inverse with (D)(V/K)(APADH) = 0.73 +/- 0.03, (D)(V/K)(Lip(S))2 = 0.77 +/- 0.03, and (D)V(app) = 0.77 +/- 0.01. Solvent deuterium kinetic isotope effects with 4,4-dithiopyridine (DTP), the 4-thiopyridone product of which requires no protonation, are also inverse with (D)(V/K)(APADH) = 0.75 +/- 0.06, (D)(V/K)(DTP) = 0.71 +/- 0.02, and (D)V(app) = 0.56 +/- 0.15. All proton inventories were linear, indicating that a single proton is being transferred in the solvent isotopically sensitive step. Taken together, these results suggest that (1) the reductive half-reaction (hydride transfer from NADH to FAD) is rate limiting when a quinone is the oxidant, and (2) deprotonation of enzymic thiols, most likely Cys(46) and Cys(41), limits the reductive and oxidative half-reactions, respectively, when D,L-lipoamide is the oxidant.
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PMID:Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes. 1156 Apr 83

Mycobacterium sp. Pyr-1 produces an enzyme with nitroreductase activity that reduces 1-nitropyrene and 4-nitrobenzoic acid to the corresponding aromatic amines. This enzyme was constitutive and required NADH; and its activity was enhanced by FAD. It was inhibited by antimycin A, dicumarol, and o-iodosobenzoic acid; and it was inactivated by ammonium sulfate precipitation. After purification to homogeneity, the protein produced a single band on native and SDS-polyacrylamide gels and had a single amino-terminal sequence. The N-terminal amino acid sequence was identical to the corresponding sequences of the lipoamide dehydrogenases of M. leprae, M. tuberculosis and Corynebacterium glutamicum. The amino-terminal sequence was also similar to lipoamide dehydrogenases from M. smegmatis and several other bacteria. The amino acid sequence of an internal peptide (12 of 13 amino acids) was nearly identical to the corresponding sequences of lipoamide dehydrogenases from M. leprae and M. tuberculosis and was similar to those of C. glutamicum, Streptomyces coelicolor and S. seoulensis. The data show that a unique lipoamide dehydrogenase in Mycobacterium sp. Pyr-1, which differs from classic (Type I) bacterial nitroreductases, reduces aromatic nitro compounds to aromatic amines.
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PMID:Purification and characterization of an enzyme from Mycobacterium sp. Pyr-1, with nitroreductase activity and an N-terminal sequence similar to lipoamide dehydrogenase. 1170 81

Submicromolar zinc inhibits alpha-ketoglutarate-dependent mitochondrial respiration. This was attributed to inhibition of the alpha-ketoglutarate dehydrogenase complex (Brown, A. M., Kristal, B. S., Effron, M. S., Shestopalov, A. I., Ullucci, P. A., Sheu, K.-F. R., Blass, J. P., and Cooper, A. J. L. (2000) J. Biol. Chem. 275, 13441-13447). Lipoamide dehydrogenase, a component of the alpha-ketoglutarate dehydrogenase complex and two other mitochondrial complexes, catalyzes the transfer of reducing equivalents from the bound dihydrolipoate of the neighboring dihydrolipoamide acyltransferase subunit to NAD(+). This reversible reaction involves two reaction centers: a thiol pair, which accepts electrons from dihydrolipoate, and a non-covalently bound FAD moiety, which transfers electrons to NAD(+). The lipoamide dehydrogenase reaction catalyzed by the purified pig heart enzyme is strongly inhibited by Zn(2+) (K(i) approximately 0.15 microm) in both directions. Steady-state kinetic studies revealed that Zn(2+) competes with oxidized lipoamide for the two-electron-reduced enzyme. Interaction of Zn(2+) with the two-electron-reduced enzyme was directly detected in anaerobic stopped-flow experiments. Lipoamide dehydrogenase also catalyzes NADH oxidation by oxygen, yielding hydrogen peroxide as the major product and superoxide radical as a minor product. Zn(2+) accelerates the oxidase reaction up to 5-fold with an activation constant of 0.09 +/- 0.02 microm. Activation is a consequence of Zn(2+) binding to the reduced catalytic thiols, which prevents delocalization of the reducing equivalents between catalytic disulfide and FAD. A kinetic scheme that satisfactorily describes the observed effects has been developed and applied to determine a number of enzyme kinetic parameters in the oxidase reaction. The distinct effects of Zn(2+) on different LADH activities represent a novel example of a reversible switch in enzyme specificity that is modulated by metal ion binding. These results suggest that Zn(2+) can interfere with mitochondrial antioxidant production and may also stimulate production of reactive oxygen species by a novel mechanism.
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PMID:Zinc is a potent inhibitor of thiol oxidoreductase activity and stimulates reactive oxygen species production by lipoamide dehydrogenase. 1174 91


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