Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa).

In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity.

A novel protein that binds to dnrN-dnrO intergenic region of Streptomyces peucetius purified by DNA affinity capture has dihydrolipoamide dehydrogenase activity.

An antitumour chemotherapeutic, daunorubicin (DNR), produced by Streptomyces peucetius exhibits cytotoxic activity through topoisomerase-mediated interaction with DNA, thereby inhibiting DNA replication and repair and RNA and protein synthesis. It is synthesized by the type II polyketide pathway. Understanding molecular mechanisms that drive expression of antibiotic biosynthetic genes in response to diverse signals and chemical inducers is of considerable interest. Intergenic DNA between regulatory genes dnrN and dnrO of DNR biosynthesis pathway in S. peucetius has a promoter for transcription of dnrN in one strand and three promoters in the opposite strand for dnrO. Studies have shown that DnrO binds to a specific sequence in this region to activate transcription of dnrN. In the present study, using biotinylated intergenic DNA in combination with streptavidin magnetic beads, we have purified a protein that binds to this target sequence. The protein has been characterized by nano LC ESI MS/MS mass spectrometry. Sequence similarity searches for effective identification of protein by genome databases comparisons led to identification of a sequence-specific DNA binding protein that exhibits dihydrolipoamide dehydrogenase (DLDH) activity suggesting that this protein may be involved in regulation of DNR biosynthesis.

Periplasmic cold expression and one-step purification of human dihydrolipoamide dehydrogenase.

Dihydrolipoamide dehydrogenase (LADH) is a FAD-linked subunit of alpha-ketoglutarate, pyruvate and branched-chain amino acid dehydrogenases and the glycine cleavage system. As an oxidoreductase it transfers electrons from the dihydrolipoic acid prosthetic group to the NAD(+) cofactor via its FAD center. Besides its physiological function it is capable of generating harmful reactive oxygen species (ROS) in pathological settings therefore it is implicated in neurodegeneration, ischemia-reperfusion, cancer and several other disorders. Pathological mutants of the enzyme cause severe, sometimes lethal syndromes like hypotonia, metabolic acidosis or inefficiency in development. Recently it has been revealed that LADH is a moonlighting protease when specific mutations in the dimerization surface destabilize the functional homodimer and expose a serine-protease-like catalytic dyad. As the basis of versatile functions of LADH is far from elucidation, there is a constant need for a pure and functional enzyme product for investigations. Several studies used recombinant human LADH before, however, it was generated by more complicated and/or physiologically less compatible protocols than reported here; most papers on functional and structural studies do not even report detailed protocols and characteristics (most importantly the purity) of their protein products. Here we describe the details of an optimized, easy-to-use periplasmic expression and one-step purification protocol for obtaining a highly pure, active and authentic (tag-cleaved) enzyme with the characterization of the protein product. The purified LADH can be used in biophysical and structural studies while the published protocol is easily convertible to a protein labeling procedure.

Lipoamide dehydrogenase from Corynebacterium glutamicum: molecular and physiological analysis of the lpd gene and characterization of the enzyme.

Lipoamide dehydrogenase (LPD) is an essential component of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, both playing a crucial role within the central metabolism of aerobic organisms. Using oligonucleotides designed according to conserved regions of LPD amino acid sequences from several organisms, the lpd gene from Corynebacterium glutamicum was identified and subsequently subcloned. The cloned lpd gene expressed in C. glutamicum cells harbouring the gene on a plasmid showed a 12-fold higher specific LPD activity when compared to the wild-type strain. DNA sequence analysis of a 4524 bp segment containing the lpd gene and adjacent regions revealed that the lpd gene is not flanked by genes encoding other subunits of the pyruvate or 2-oxoglutarate dehydrogenase complexes and predicted an LPD polypeptide of 469 amino acids with an M(r) of 50619. The amino acid sequence of this polypeptide shows between 26 and 58% identity when compared to LPD enzymes from other organisms. Transcriptional analyses revealed that the lpd gene from C. glutamicum is monocistronic (1.45 kb mRNA) and that its transcription is initiated exactly at the nucleotide defined as the translational start. LPD was purified and biochemically characterized. This analysis revealed that the enzyme catalyses the reversible reoxidation of dihydrolipoic acid and NADH:NAD(+) transhydrogenation, and is able to transfer electrons from NADH to various redox-active compounds and quinones. An in vivo participation of C. glutamicum LPD in facilitation of quinone redox cycling is proposed.

Subunit interactions in the mammalian alpha-ketoglutarate dehydrogenase complex. Evidence for direct association of the alpha-ketoglutarate dehydrogenase and dihydrolipoamide dehydrogenase components.

Selective tryptic proteolysis of the mammalian alpha-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and also a fully active E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E3-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the specific cross-reactivities of subunit-specific antisera. Analysis of the pattern of release of E1 and E1' polypeptides from the OGDC during tryptic inactivation suggests that both polypeptide chains of individual E1 homodimers must be cleaved to permit the dissociation of the E1 and E3 components. A new protocol has been devised that promotes E1 dissociation from the oligomeric dihydrolipoamide succinyltransferase (E2) core in an active state. Significant levels of overall OGDC reconstitution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.

Identification and characterization of the acoD gene encoding a dihydrolipoamide dehydrogenase of the Klebsiella pneumoniae acetoin dehydrogenase system.

The acoD gene, which encodes a dihydrolipoamide dehydrogenase component of the acetoin dehydrogenase enzyme system of Klebsiella pneumoniae was isolated and the nucleotide sequence determined. The gene is capable of encoding a protein of 465 amino acid residues with conserved binding domains for NAD and FAD, and two redox-active cysteine residues. The acoD gene product exhibited a Michaelis constant of 170 microM for NAD, while NADP can not be used as a substrate. The purified enzyme appeared to be a dimer of the acoD gene product. It did not associate tightly with the E1 and E2 components of either acetoin dehydrogenase or 2-oxoglutarate dehydrogenase to form an active multi-enzyme complex.

Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells.

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.

Biochemical and molecular characterization of the Clostridium magnum acetoin dehydrogenase enzyme system.

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi.

From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.

Immunological identification of a new component of Ascaris pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex was purified from Ascaris muscle both with and without MgCl2 treatment at the first stage of purification. The specific activity of complex purified with MgCl2 treatment was about 2-fold as high as that purified without it. In addition to three component enzymes, two unknown polypeptides of 46 and 41 kDa were found in the complex purified by the two procedures. The quantity of unknown polypeptide of 41 kDa was increased in the complex purified with MgCl2 treatment as compared with that without it. Antibodies against the three component enzymes were prepared. All the antibodies precipitated the two unknown polypeptides in addition to the three component enzymes in immunoprecipitation experiments. Antibody against the alpha-subunit of pyruvate dehydrogenase reacted with the 41 kDa polypeptide as well as the alpha-subunit in the immunoblotting method. The unknown polypeptide of 46 kDa did not react with any antibody. These results suggest that the unknown 41 kDa polypeptide is a derivative of the alpha-subunit and that the unknown 46 kDa polypeptide is not a proteolytic-degradative product of component enzymes but is a component of the Ascaris pyruvate dehydrogenase complex. When the Ascaris complex was incubated with [2-14C]pyruvate in the absence of CoASH, only lipoate acetyltransferase was acetylated. In rat heart pyruvate dehydrogenase complex, lipoate acetyltransferase and another protein (referred to as component x or protein x) were acetylated. These results indicate that the unknown polypeptide of 46 kDa is a new component.

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex.

Pyruvate dehydrogenase complex was purified from rat heart. The complex showed four polypeptide bands on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, corresponding to lipoate acetyltransferase (mol.wt. 68 000), lipoamide dehydrogenase (mol.wt. 56 000), alpha-subunit (mol.wt. 41 000) and beta-subunit (mol.wt. 35 000) of pyruvate dehydrogenase. Rat heart pyruvate dehydrogenase complex was dissociated into three component enzymes and the antibodies against each component enzyme were prepared. Anti-pyruvate dehydrogenase and anti-lipoate acetyltransferase antibodies effectively precipitated pyruvate dehydrogenase complex, but an anti-lipoamide dehydrogenase antibody released lipoamide dehydrogenase from the complex and effectively precipitated lipoamide dehydrogenase. Lipoamide dehydrogenase was synthesized in a cell-free reticulocyte lysate system with total RNA from rat liver. Its translation product was detected as a putative precursor which is 3000 Da larger than the mature subunit. In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in free polysomes than in membrane-bound polysomes.

Wild-type and mutant forms of the pyruvate dehydrogenase multienzyme complex from Bacillus subtilis.

A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

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.

Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from Escherichia coli.

The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.

Subunit structure of dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex from bovine kidney.

The pyruvate dehydrogenase complex has been isolated from bovine kidney mitochondria under special anti-proteolytic conditions yielding preparations with a specific activity of up to 20 U/mg protein. Dihydrolipoamide acetyltransferase resolved from the complex was subjected to limited proteolysis resulting in the formation of two major fragments with apparent molecular weights of 36000 and 28000. The fragments were isolated by extraction from dodecyl sulfate polyacrylamide gels and were both shown to possess enzymatic activity for acetyl transfer. Acetylation studies indicated that each fragment contains one protein-bound lipoyl group. It is concluded that the kidney dihydrolipoamide acetyltransferase subunit consists of two homologous if not identical domains. A model is suggested where the acetyltransferase core of the mammalian pyruvate dehydrogenase complex is made up of 30 polypeptide chains whose 60 domains could be arranged in pentagonal dodecahedron symmetry quite similar as proposed for the 60 subunit structure of the acetyltransferase core.

[Isolation and characterization of a membrane-bound pyruvate dehydrogenase complex from the phototrophic bacterium Rhodospirillum rubrum (author's transl)]. / Isolierung und Charakterisierung eines membrangebundenen Pyruvatdehydrogenase-Komplexes aus dem phototrophen Bakterium Rhodospirillum rubrum.

The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhodospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5-2mumol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R. rubrum revealed no cooperatively between pyruvate binding sites. The kinetic constants (Km) for the overall reaction were (in mM): 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 micrometer. NADH was a potent inhibitor (Ki=5 micrometer) of the enzyme complex and the dihydrolipo amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki=40 micrometer). The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mM) had no pronounced effect on the overall reaction.