Dihydrolipoamide dehydrogenase from Leishmania donovani: New insights through biochemical characterization.

Dihydrolipoamide dehydrogenase (DLDH) regulates many crucial metabolic pathways as a multi-enzyme complex. Leishmania donovani dihydrolipoamide dehydrogenase (LdDLDH) has two variants present on two different chromosomes with very less sequence similarities. In the current study, we cloned both the variants in pET28a (+) vector and expressed in Rosetta-gami (DE3) E. coli strain. Expressed proteins were finally purified from pellets using Ni-NTA affinity chromatography. Purified enzymes were biochemically characterized and different kinetic parameters were studied. Both the variants showed maximum activity in pH range of 7.0-8.0 and temperature 50±5°C in the physiological direction. The estimated Km for dihydrolipoamide (DLA) and NAD+ were 2.7±0.48mM and 171.23±11.59µM respectively for variant 1 (LdBPK291950.1). In the case of variant 2 (LdBPK323510.1), Km values for DLA and NAD+ were found to be 829.85±37µM and 226±1.56µM respectively. The variant 2 was more efficient in terms of activity. While both the forms of the enzymes showed diaphorase activity, variant 1 was found to be better. Sequence dissimilarities of both forms were analyzed for biological insights.

Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 µM and 0.37 µmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification.

Biochemical and structural characterization of the apicoplast dihydrolipoamide dehydrogenase of Plasmodium falciparum.

PDC (pyruvate dehydrogenase complex) is a multi-enzyme complex comprising an E1 (pyruvate decarboxylase), an E2 (dihydrolipomide acetyltransferase) and an E3 (dihydrolipoamide dehydrogenase). PDC catalyses the decarboxylation of pyruvate and forms acetyl-CoA and NADH. In the human malaria parasite Plasmodium falciparum, the single PDC is located exclusively in the apicoplast. Plasmodium PDC is essential for parasite survival in the mosquito vector and for late liver stage development in the human host, suggesting its suitability as a target for intervention strategies against malaria. Here, PfaE3 (P. falciparum apicoplast E3) was recombinantly expressed and characterized. Biochemical parameters were comparable with those determined for E3 from other organisms. A homology model for PfaE3 reveals an extra anti-parallel ß-strand at the position where human E3BP (E3-binding protein) interacts with E3; a parasite-specific feature that may be exploitable for drug discovery against PDC. To assess the biological role of Pfae3, it was deleted from P. falciparum and although the mutants are viable, they displayed a highly synchronous growth phenotype during intra-erythrocytic development. The mutants also showed changes in the expression of some mitochondrial and antioxidant proteins suggesting that deletion of Pfae3 impacts on the parasite's metabolic function with downstream effects on the parasite's redox homoeostasis and cell cycle.

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.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Breaking Caenorhabditis elegans the easy way using the Balch homogenizer: an old tool for a new application.

The nematode Caenorhabditis elegans is a model organism best known for its powerful genetics. There is an increasing need in the worm community to couple genetics with biochemistry. Isolation of functionally active proteins or nucleic acids without the use of strong oxidizing denaturants or of subcellular compartments from C. elegans has, however, been challenging because of the worms' thick surrounding cuticle. The Balch homogenizer is a tool that has found much use in mammalian cell culture biology. The interchangeable single ball-bearing design of this instrument permits rapid permeabilization, or homogenization, of cells. Here we demonstrate the utility of the Balch homogenizer for studies with C. elegans. We describe procedures for the efficient breakage and homogenization of every larval stage, including dauers, and show that the Balch homogenizer can be used to extract functionally active proteins. Enzymatic assays for catalase and dihydrolipoamide dehydrogenase show that sample preparation using the Balch homogenizer equals or outperforms conventional methods employing boiling, sonication, or Dounce homogenization. We also describe phenol-free techniques for isolation of genomic DNA and RNA. Finally, we used the tool to isolate coupled mitochondria and polysomes. The reusable Balch homogenizer represents a quick and convenient solution for undertaking biochemical studies on C. elegans.

Molecular identification of the enzyme responsible for the mitochondrial NADH-supported ammonium-dependent hydrogen peroxide production.

A homogeneous protein with a subunit apparent molecular mass of ∼50 kDa that catalyzes the previously described mitochondrial NADH-supported ammonium-stimulated hydrogen peroxide production (Grivennikova, V.G., Gecchini, G. and Vinogradov, A.D. (2008) FEBS Lett. 583, 1287-1291) was purified from the mitochondrial matrix of bovine heart. Chromatography of partially purified protein showed that the peaks of ammonium-stimulated NADH-dependent H(2)O(2) production and that of NADH:lipoamide oxidoreductase activity coincided. The catalytic properties and mass spectrometry of the trypsin-digested protein revealed peptides that allowed identification of the protein as the Bos taurus dihydrolipoyl dehydrogenase.

Cloning and purification of recombinant silkworm dihydrolipoamide dehydrogenase expressed in Escherichia coli.

Dihydrolipoamide dehydrogenase (DLDH), a flavin-dependent oxidoreductase is essential for energy metabolism. As an oxidoreductase it catalyzes the NAD(+)-dependent oxidation of dihydrolipoamide. In this study, a putative Bombyx mori dihydrolipoamide dehydrogenase (BmDLDH) gene was cloned, expressed, purified and characterized for the first time. The BmDLDH gene was amplified from a pool of silkworm cDNAs by PCR and cloned into Escherichia coli expression vector pET-28a(+). The recombinant His-tagged BmDLDH protein was expressed in E. coli BL21 (DE3) and purified by metal chelating affinity chromatography. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis. Furthermore, the oxidoreductase activity in the reverse reaction indicated that the soluble recombinant BmDLDH produced at lower growth temperature was able to catalyze the lipoamide-dependent oxidation of NADH.

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.

The dihydrolipoamide dehydrogenase from the crenarchaeon Acidianus ambivalens.

A dihydrolipoamide dehydrogenase (DLDH) was purified and characterized for the first time from a crenarchaeon, Acidianus ambivalens. The holoenzyme consists of two identical subunits with a molecular mass of 45.4 kDa per monomer. It contains FAD as a prosthetic group and uses NAD+ as the preferential substrate, but can also reduce NADP+. The Michaelis-Menten constants of the forward (NAD+ reduction) and reverse (NADH oxidation) reactions were KM (dihydrolipoamide)=0.70 mM, KM (NAD+)=0.71 mM, KM (lipoamide)=1.26 mM and KM (NADH)=3.15 microM. A comparative study of NADH:lipoamide oxidoreductase and NADH:K3[Fe(CN)6] oxidoreductase activities was performed, the optimal temperature and pH being different for each: 55 degrees C, pH 7 and 89 degrees C, pH 5.5, respectively. Although DLDH is generally part of the alpha-ketoacid dehydrogenase complexes in Bacteria and Eukarya, none of these complexes has yet been isolated from Sulfolobales. The metabolic role of DLDH in these organisms is discussed.

Dihydrolipoamide dehydrogenase from porcine heart catalyzes NADH-dependent scavenging of nitric oxide.

Dihydrolipoamide dehydrogenase (DLDH; EC 1.8.1.4) from porcine heart is capable of using nitric oxide (NO) as an electron acceptor, with NADH as the electron donor, forming nitrate in the reaction. NADPH was not effective as an electron donor. The reaction had a pH optimum near 6 and was not inhibited by cyanide or diphenyleneiodonium ions. The Km for NADH was 10 microM, while that for NO was 0.5 microM. The rate of NO conversion was comparable to the rate of lipoamide conversion (200 micromol min(-1) mg(-1) protein at pH 6). Cytochrome c or myoglobin were poor electron acceptors by themselves but, in the presence of methylene blue, DLDH had an activity of 5-7 micromol min(-1) mg(-1) protein with these substrates, indicating that DLDH can act also as a methemoglobin reductase. While the Km of DLDH for NO is relatively low, it is in the physiological range of NO levels encountered in the tissue. The enzyme may, therefore, have a significant role in modifying NO levels under specific cell conditions.

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase.

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.

Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for reductive acetylation of dihydrolipoamide acetyltransferase.

Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.

Molecular cloning, functional characterization, and subcellular localization of soybean nodule dihydrolipoamide reductase.

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

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.

Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the formation of a precatalytic complex with 2-oxoglutarate.

The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.

Enhanced sensitivity of Streptomyces seoulensis to menadione by superfluous lipoamide dehydrogenase.

Lipoamide dehydrogenase from Streptomyces seoulensis could facilitate menadione-mediated cytochrome c reduction, which was mostly inhibited by superoxide dismutase, indicating the obvious involvement of superoxide radical anion. In this reaction, the production of superoxide radical anion occurred via a menadione semiquinone radical anion. When exposed to menadione, lipoamide dehydrogenase-overexpressing cells showed a much lower survival rate with a concomitant decrease of intracellular protein thiol than the wild-type strain. These results suggest that lipoamide dehydrogenase is a facilitating agent in the redox cycling of quinone compounds in vivo as well as in vitro and could inevitably increase the potential toxicity of the compounds.

Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route.

Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.

Expression, reactivation, and purification of enzymes from Haloferax volcanii in Escherichia coli.

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.