Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.     

Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate

Dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase were formed when Pelobacter carbinolicus strain GraBd1 was grown on acetoin. The specific activities of these enzymes amounted to 0.50 and 28.7 U/mg protein, respectively. The crude extract catalyzed the CoASH- and NAD+-dependent formation of acetyl-CoA from acetoin and methylacetoin. From ethylene glycol-grown cells these activities were absent. Crude extracts also exhibited acetoin: methyl viologen and acetoin: metronidazole oxidoreductase activity. As shown by reconstitution experiments methylviologen reduction was dependent on the presence of a light-brownish protein (Mr 220,000 +/- 10,000); metronidazole reduction was in addition dependent on the presence of a dark-brownish protein (Mr 4,900 +/- 800), which is probably a ferredoxin. However, both components were synthesized constitutively. We discussed a model for oxidative-thiolytic cleavage of acetoin which is analogous to the reaction of the pyruvate dehydrogenase enzyme complex rather than to pyruvate: ferredoxin oxidoreductase.     

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.     

Crystal structures of the key anaerobic enzyme pyruvate:ferredoxin oxidoreductase, free and in complex with pyruvate

Oxidative decarboxylation of pyruvate to form acetyl-coenzyme A, a crucial step in many metabolic pathways, is carried out in most aerobic organisms by the multienzyme complex pyruvate dehydrogenase. In most anaerobes, the same reaction is usually catalyzed by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFOR). Thus, PFOR is a potential target for drug design against certain anaerobic pathogens. Here, we report the crystal structures of the homodimeric Desulfovibrio africanus PFOR (data to 2.3 A resolution), and of its complex with pyruvate (3.0 A resolution). The structures show that each subunit consists of seven domains, one of which affords protection against oxygen. The thiamin pyrophosphate (TPP) cofactor and the three [4Fe-4S] clusters are suitably arranged to provide a plausible electron transfer pathway. In addition, the PFOR-pyruvate complex structure shows the noncovalent fixation of the substrate before the catalytic reaction.     

CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase.

CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin.     

Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.     

Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 +/- 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.     

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.     

Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.     

Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium

Pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase were obtained from cell-free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting-out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of non-identical subunits of Mr 86000 and 42000 in the case of the pyruvate-degrading enzyme and of 88000 and 36000 in the case of the 20 -oxogluatarate-degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe-4S]2 + (2 + , 1+) clusters and two molecules of thiamin diphosphate. Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2-oxoacid:ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2-oxoacid dehydrogenase multienzyme complexes which commonly occur in anaerobic organisms.     

Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus

A low molecular weight iron-sulfur protein has been purified from Tritrichomonas foetus by deoxycholate extraction of whole cells, ion exchange chromatography, and gel filtration. The purified protein was essentially homogeneous as judged by isoelectric focusing, polyacrylamide gel electrophoresis, and gel filtration. A pI of 4.3 was observed. The molecular weight of the protein was estimated to be 12,000. Chemical and spectral analysis showed the protein to have a [2Fe-2S] cluster. The absorbance spectrum of the oxidized protein showed maxima at 280, 340, 458 and shoulders at 410 and 550 nm. The maximum observed A458/A280 ratio was 0.82 and the absorbance of the oxidized protein at 458 nm was 8,000 M-1 X cm-1. The low temperature EPR spectrum of the protein reduced with dithionite revealed axial symmetry with features at g values of g = 1.94 and g = 2.02. The oxidized protein gave no EPR signal in the g = 1.8 to 2.2 range. Cell fractionation studies indicated the localization of this protein in the hydrogenosome. The protein was able to function as an electron transport component in the reduction of metronidazole (a 5-nitroimidazole derivative) by pyruvate:ferredoxin oxidoreductase and hydrogenase from T. foetus and also from Trichomonas vaginalis and Clostridium pasteurianum as well as in the reduction of cytochrome c by plant NADPH:ferredoxin oxidoreductase. This protein has the characteristics of a ferredoxin and is likely to be a physiological electron carrier in hydrogenosomal pyruvate oxidation.     

Purification and characterization of Haemophilus influenzae D-lactate dehydrogenase

Haemophilus influenzae D(-)-lactate dehydrogenase (D(-)-lactate:NAD oxidoreductase; EC 1.1.1.28) was purified to electrophoretic homogeneity using salt fractionation, hydrophobic and dye affinity chromatography. The enzyme was purified 2100-fold with a 14% recovery and a final specific activity of 300 units/mg protein. The enzyme was demonstrated to be a tetramer of Mr 135,000. The enzyme catalyzed the reduction of pyruvate to give exclusively D(-)-lactate using NADH as coenzyme. The reaction catalyzed was essentially unidirectional, with the oxidation of D-lactate in the presence of NAD proceeding at less than 0.2% the rate of pyruvate reduction. Kinetic parameters for the reduction of pyruvate were determined for NADH and four structural analogs of the coenzyme. Coenzyme-competitive inhibition by adenosine derivatives indicated the presence of regions in the coenzyme binding site interacting with the adenosine and pyrophosphate moieties of the coenzyme. The purified enzyme was sensitive to oxidation and was effectively inactivated by sulfhydryl reagents. Conversion of D-lactate to pyruvate catalyzed by a membrane-bound D-lactate oxidase was demonstrated in cell-free extracts of H. influenzae.     

Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition,catalytic properties,and sequence alignments

Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon. In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism. The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16%. The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating and structure. Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur. FAD, FMN, lipoic acid, and copper were not found. The purified enzyme showed an apparent K _m for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent V _max of 64 U/mg (at 65°C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70°C). The temperature optimum was above 90°C. At 90°C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl. The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate. The N-terminal amino acid sequences of the four subunits were determined. The sequence of the -subunit had similarities to the N-terminal amino acid sequence of the -subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate: ferredoxin oxidoreductase from proteobacteria and from cyanobacteria. No sequence similarities were found, however, between the -subunits of the enzyme from A. fulgidus and the heterodimeric pyruvate: ferredoxin oxidoreductase from Halobacterium halobium.Abbreviations CoASH Coenzyme A - F ₄₂₀ Coenzyme F₄₂₀     

Metabolism in hyperthermophilic microorganisms

Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H₂S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H₂ and CO₂, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO₂ do so via a reductive citric acid cycle. Two S^o-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S^o. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S^o has been proposed. However, the mechanisms by which S^o reduction is coupled to energy conservation in this organism and in obligate S^o-reducing hyperthermophiles is not known.Abbreviations ADH alcohol dehydrogenase (ADH) - AOR aldehyde ferredoxin oxidoreductase - FMOR formate ferredoxin oxidoreductase - FOR formaldehyde ferredoxin oxidoreductase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - GluOR glucose ferredoxin oxidoreductase - KGOR 2-ketoglutarate ferredoxin oxidoreductase - IOR indolepyruvate ferredoxin oxidoreductase - LDH lactate dehydrogenase - MPT molybopterin - POR pyruvate ferredoxin oxidoreductase - PLP pyridoxal-phosphate - PS polysulfide - TPP thiamin pyrophosphate - S^o elemental sulfur - VOR isovalerate ferredoxin oxidoreductase     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Primary structure and eubacterial relationships of the pyruvate:Ferredoxin oxidoreductase of the amitochondriate eukaryoteTrichomonas vaginalis

In the eukaryotic unicellular organismTrichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex. This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane. PFO and its closely related homolog, pyruvate: flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria. The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups. To gain more insight into the evolutionary relationships ofT. vaginalis PFO we determined the primary structure of its two genes (pfoA andpfoB). The deduced amino acid sequences showed 95% positional identity. Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved. TheT. vaginalis PFOs were found to be homologous to eubacterial pyruvate: flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length. Lack of eubacterial PFO sequences precluded a comparison.pfoA andpfoB revealed a greater distance from related enzymes of Archaebacteria. The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein. This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins. These sequences are assumed to be involved in organellar targeting and import. The results underscore the unusual characteristics ofT. vaginalis metabolism and of their hydrogenosomes. They also suggest that in its energy metabolismT. vaginalis is closer to eubacteria than archaebacteria.Abbreviations PCR DNA polymerase chain reaction - PDH pyruvate dehydrogenase - PFO pyruvate:ferredoxin oxidoreductase - TPP thiamine pyrophosphate Correspondence to: M. Müller     

Coenzyme-binding sites of the brain pyruvate dehydrogenase complex]

It is shown that the relative amount of the holoenzyme in the highly purified pyruvate dehydrogenase complex from the bovine brain is higher when the enzyme activity is assayed in the reaction of nonoxidative formation of acetaldehyde as compared to the pyruvate: NAD+ reductase reaction. The S0.5 values for thiamine pyrophosphate are as following: (TPP) (0.314 +/- 0.22) x 10(-7) M with reaction of nonoxidative formation of acetaldehyde, (0.188 +/- 0.08) x 10(-6) M and (1.65 +/- 1.16) x 10(-6) M in case of the pyruvate: NAD+ reductase reaction. TPP in the concentration of (0.5-6.0) x 10(-7) M completely protects the sites of nonoxidative formation of acetaldehyde from modification by the coenzyme analogs, 4'-oxythiamine pyrophosphate and tetrahydrothiamine pyrophosphate. However, the pyruvate: NAD+ reductase activity of the pyruvate dehydrogenase complex is inhibited in this case by 30-34%. The data obtained suggest that in contrast to the pyruvate: NAD+ reductase reaction the conversion of pyruvate to acetaldehyde occurs by the sites which tightly bound TPP.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.