RESUMEN

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T. profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.

Asunto(s)

Cetona Oxidorreductasas/metabolismo , Thermococcus/enzimología , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Semivida , Calor , Hierro/metabolismo , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Peso Molecular , Oxígeno/farmacología , Azufre/metabolismo , Thermococcus/efectos de los fármacos

RESUMEN

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.

Asunto(s)

Aflatoxinas/biosíntesis , Alcohol Deshidrogenasa/genética , Cetona Oxidorreductasas/genética , Levilactobacillus brevis/enzimología , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/aislamiento & purificación , Secuencia de Aminoácidos , Antraquinonas/metabolismo , Catálisis , Clonación Molecular , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Datos de Secuencia Molecular

RESUMEN

A hyperthermophilic and aerobic crenarchaeon, Aeropyrum pernix K1, has two sets of genes possibly encoding 2-oxoacid:ferredoxin oxidoreductases. One is encoded in open reading frames (ORFs) ape2126 and ape2128, and the other in ORFs ape1473 and ape1472. The two sets of genes were expressed. The product enzymes, Ape2126/2128 and Ape1473/1472, showed optimal temperatures of 105 and over 110 degrees C, and optimal pHs of 8.5 and 9.0, respectively, using pyruvate as a substrate. Pyruvate, 2-oxobutyrate, and glyoxylate were the best substrates for both enzymes, and additionally Ape1473/1472 was able to act on 2-oxoglutarate, suggesting the enzyme operates in the TCA cycle.

Asunto(s)

Aeropyrum/enzimología , Aeropyrum/genética , Regulación de la Expresión Génica Arqueal , Regulación Enzimológica de la Expresión Génica , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/metabolismo , Aeropyrum/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Ácido Pirúvico/metabolismo , Especificidad por Sustrato , Temperatura

RESUMEN

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).

Asunto(s)

Aminoácidos/metabolismo , Metabolismo Energético/fisiología , Proteínas Hierro-Azufre/metabolismo , Cetona Oxidorreductasas/metabolismo , Thermococcus/enzimología , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/aislamiento & purificación , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo

RESUMEN

L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis.

Asunto(s)

Desoxiazúcares/biosíntesis , Azúcares de Guanosina Difosfato/metabolismo , Cetona Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clonación Molecular , Deuterio/metabolismo , Electroforesis en Gel de Poliacrilamida , Hexosiltransferasas/genética , Hexosiltransferasas/aislamiento & purificación , Hexosiltransferasas/metabolismo , Hidrógeno/metabolismo , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/aislamiento & purificación , Cinética , NAD/metabolismo , Yersinia pseudotuberculosis/enzimología , Yersinia pseudotuberculosis/genética

RESUMEN

In autotrophic methanogens, pyruvate oxidoreductase (POR) plays a key role in the assimilation of CO(2) and the biosynthesis of organic carbon. This enzyme has been purified to homogeneity, and the genes from Methanococcus maripaludis were sequenced. The purified POR contained five polypeptides with molecular masses of 47, 33, 25, 21.5 and 13 kDa. The N-terminal sequences of four of the polypeptides had high similarity to the subunits commonly associated with this enzyme from other archaea. However, the 21.5-kDa polypeptide had not been previously observed in PORs. Nucleotide sequencing of the gene cluster encoding the POR revealed six open reading frames ( porABCDEF). The genes porABCD corresponded to the subunits previously identified in PORs. On the basis of the N-terminal amino acid sequence, porE encoded the 21.5-kDa polypeptide and contained a high cysteinyl residue content and a motif indicative of a [Fe-S] cluster. porF also had a high sequence similarity to porE, a high cysteinyl residue content, and two [Fe-S] cluster motifs. Homologs to porE were also present in the genomic sequences of the autotrophic methanogens Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus. Based upon these results, it is proposed that PorE and PorF are components of a specialized system required to transfer low-potential electrons for pyruvate biosynthesis. Some biochemical properties of the purified methanococcal POR were also determined. This unstable enzyme was very sensitive to O(2 )and demonstrated high activity with pyruvate, oxaloacetate, and alpha-ketobutyrate. Methyl viologen, rubredoxin, FMN, and FAD were readily reduced. Activity was also observed with spinach and clostridial ferredoxins and cytochrome c. Coenzyme F(420) was not an electron acceptor for the purified enzyme.

Asunto(s)

Cetona Oxidorreductasas/metabolismo , Methanococcus/enzimología , Secuencia de Aminoácidos , Catálisis , Ditionita/metabolismo , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/aislamiento & purificación , Methanococcus/citología , Datos de Secuencia Molecular , Filogenia , Piruvato-Sintasa , Homología de Secuencia de Aminoácido , Temperatura

Asunto(s)

Cetona Oxidorreductasas/aislamiento & purificación , Cetona Oxidorreductasas/metabolismo , Pyrococcus furiosus/enzimología , Cromatografía/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Dimerización , Durapatita , Cetona Oxidorreductasas/química , Cinética , Subunidades de Proteína , Piruvato-Sintasa , Especificidad por Sustrato

RESUMEN

The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/enzimología , Fucosa/análogos & derivados , Fucosa/biosíntesis , Glicósidos/biosíntesis , Guanosina Difosfato Fucosa/síntesis química , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/aislamiento & purificación , Carbohidrato Epimerasas/metabolismo , Cationes Bivalentes/farmacología , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/genética , Fucosa/aislamiento & purificación , Guanosina Difosfato Manosa/análogos & derivados , Guanosina Difosfato Manosa/metabolismo , Concentración de Iones de Hidrógeno , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/aislamiento & purificación , Cetona Oxidorreductasas/metabolismo , Cinética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificación , Deshidrogenasas del Alcohol de Azúcar/metabolismo

Asunto(s)

Cetona Oxidorreductasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación , Pseudomonas/enzimología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Liquida , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética

RESUMEN

BkdR can be isolated in nearly pure form as a tetramer by this procedure, which involves hyperexpressing bkdR from a plasmid, purification by chromatography on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, and dialysis to precipitate BkdR. BkdR is relatively insoluble in aqueous buffers but can be kept in solution in buffer with 50% (v/v) glycerol and 0.2 M NaCl. Cultures of E. coli DH5 alpha (pJRS119) should be maintained at 30 degrees to promote plasmid stability. Because BkdR is prone to form intermolecular disulfide bonds, buffers for SDS-PAGE should contain fresh 0.5% (v/v) 2-mercaptoethanol.

Asunto(s)

Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , Pseudomonas/enzimología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cetona Oxidorreductasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación

Asunto(s)

Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/enzimología , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cetona Oxidorreductasas/aislamiento & purificación , Complejos Multienzimáticos/aislamiento & purificación

RESUMEN

Branched-chain alpha-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1beta and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.

Asunto(s)

Arabidopsis/enzimología , ADN Complementario/metabolismo , Cetona Oxidorreductasas/metabolismo , Complejos Multienzimáticos/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Envejecimiento , Secuencia de Aminoácidos , Diurona/farmacología , Expresión Génica , Biblioteca de Genes , Herbicidas/farmacología , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/aislamiento & purificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Sacarosa/farmacología , Factores de Tiempo , Distribución Tisular

RESUMEN

Phenylacetic acids are common intermediates in the microbial metabolism of various aromatic substrates including phenylalanine. In the denitrifying bacterium Thauera aromatica phenylacetate is oxidized, under anoxic conditions, to the common intermediate benzoyl-CoA via the intermediates phenylacetyl-CoA and phenylglyoxylate (benzoylformate). The enzyme that catalyzes the four-electron oxidation of phenylacetyl-CoA has been purified from this bacterium and studied. The enzyme preparation catalyzes the reaction phenylacetyl-CoA + 2 quinone + 2 H2O --> phenylglyoxylate + 2 quinone H2 + CoASH. Phenylacetyl-CoA:acceptor oxidoreductase is a membrane-bound molybdenum-iron-sulfur protein. The purest preparations contained three subunits of 93, 27, and 26 kDa. Ubiquinone is most likely to act as the electron acceptor, and the oxygen atom introduced into the product is derived from water. The protein preparations contained 0.66 mol Mo, 30 mol Fe, and 25 mol acid-labile sulfur per mol of native enzyme, assuming a native molecular mass of 280 kDa. Phenylglyoxylyl-CoA, but not mandelyl-CoA, was observed as a free intermediate. All enzyme preparations also catalyzed the subsequent hydrolytic release of coenzyme A from phenylglyoxylyl-CoA but not from phenylacetyl-CoA. The enzyme is reversibly inactivated by a low concentration of cyanide, but is remarkably stable with respect to oxygen. This new member of the molybdoproteins represents the first example of an enzyme which catalyzes the alpha-oxidation of a CoA-activated carboxylic acid without utilizing molecular oxygen.

Asunto(s)

Bacterias Gramnegativas/enzimología , Cetona Oxidorreductasas/metabolismo , Proteínas de la Membrana/metabolismo , Fenilalanina/metabolismo , Secuencia de Aminoácidos , Catálisis , Sistema Libre de Células , Cromatografía Líquida de Alta Presión , Cianuros/farmacología , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Datos de Secuencia Molecular

RESUMEN

We have induced high levels of resistance to metronidazole (1 mM or 170 microg ml(-1)) in two different strains of Trichomonas vaginalis (BRIS/92/STDL/F1623 and BRIS/92/STDL/B7708) and have used one strain to identify two alternative T. vaginalis 2-keto acid oxidoreductases (KOR) both of which are distinct from the already characterised pyruvate:ferredoxin oxidoreductase (PFOR). Unlike the characterised PFOR which is severely down-regulated in metronidazole-resistant parasites, both of the alternative KORs are fully active in metronidazole-resistant T. vaginalis. The first, KORI, localized in all membrane fractions but predominantly in the hydrogenosome fraction, is soluble in Triton X-100 and the second, KOR2, is extractable in 1 M acetate from membrane fractions of metronidazole-resistant parasites. PFOR and both KORI and KOR2 use a broad range of 2-keto acids as substrates (pyruvate, alpha-ketobutyrate, alpha-ketomalonate), including the deaminated forms of aromatic amino acids (indolepyruvate and phenylpyruvate). However, unlike PFOR neither KORI or KOR2 was able to use oz-ketoglutarate. Deaminated forms of branched chain amino acids (alpha-ketoisovalerate) were not substrates for T. vaginalis KORs. Since KOR I and KOR2 do not apparently donate electrons to ferredoxin, and are not down-regulated in metronidazole-resistant parasites, we propose that KORI and KOR2 provide metronidazole-resistant parasites with an alternative energy production pathway(s) which circumvents metronidazole activation.

Asunto(s)

Antitricomonas/farmacología , Cetona Oxidorreductasas/aislamiento & purificación , Metronidazol/farmacología , Trichomonas vaginalis/enzimología , Animales , Compartimento Celular , Resistencia a Medicamentos , Metabolismo Energético , Isoenzimas , Cetona Oxidorreductasas/biosíntesis , Cetona Oxidorreductasas/genética , Piruvato-Sintasa , ARN Mensajero/aislamiento & purificación , ARN Protozoario/aislamiento & purificación , Solubilidad , Fracciones Subcelulares/enzimología

RESUMEN

For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.

Asunto(s)

Desulfovibrio/enzimología , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Metabolismo Energético , Cetona Oxidorreductasas/metabolismo , Piruvato-Sintasa

RESUMEN

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

Asunto(s)

Evolución Molecular , Proteínas Hierro-Azufre/metabolismo , Cetona Oxidorreductasas/metabolismo , Pyrococcus/enzimología , Secuencia de Aminoácidos , Transporte de Electrón , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/aislamiento & purificación , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/aislamiento & purificación , Espectrometría de Masas , Datos de Secuencia Molecular , Oxidación-Reducción , Pyrococcus/genética , Piruvato-Sintasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Espectrofotometría Ultravioleta

RESUMEN

This work investigated the cellular location of the pyruvate:ferredoxin oxidoreductase (PFO) enzyme in Entamoeba. A 1.9 kb fragment located at the 3' end of the Ehpfo gene was cloned in the pRSETB vector and expressed. The recombinant peptide was purified and inoculated in rabbits. By Western blot assays the antibodies detected a single 130 kDa band in all E. histolytica strains tested and in E. moshkovskii. By immunofluorescence, the antibodies showed the presence of PFO in the plasma membrane and in a cytoplasmic structure that appeared as a ring or as a compact small body in E. histolytica strains. In E. invadens and E. moshkovskii (strains FIC and Laredo) PFO was located in the plasma membrane showing different fluorescence patterns. Immunofluorescence on E. histolytica synchronized cultures showed that the cytoplasmic structure appeared in 85, 60, 20 and 10% of the trophozoites in mitosis, G1, S and G2 phases, respectively. By in situ hybridization the Ehpfo gene was found in the nuclei and the trophozoites of the clone A, strain HM1:IMSS, differed in the Ehpfo gene content.

Asunto(s)

Entamoeba/enzimología , Cetona Oxidorreductasas/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Animales , Anticuerpos Antiprotozoarios , División Celular , Membrana Celular/enzimología , Clonación Molecular , Entamoeba/clasificación , Entamoeba/citología , Piruvato-Sintasa , Conejos , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Anaerobic oxidation of phenylalanine and phenylacetate proceeds via alpha-oxidation of phenylacetyl-CoA to phenylglyoxylate. This four-electron oxidation system was studied in the denitrifying bacterium Thauera aromatica. It is membrane-bound and was solubilized with Triton X-100. The system used dichlorophenolindophenol as an artificial electron acceptor; a spectrophotometric assay was developed. No other products besides phenylglyoxylate and coenzyme A were observed. The enzyme was quite oxygen-insensitive and was inactivated by low concentrations of cyanide. Enzyme activity was induced under denitrifying conditions with phenylalanine and phenylacetate, it was low in cells grown with phenylglyoxylate, and it was virtually absent in cells grown with benzoate and nitrate or after aerobic growth with phenylacetate.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Bacilos Gramnegativos Anaerobios Facultativos/enzimología , Cetona Oxidorreductasas/aislamiento & purificación , Anaerobiosis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Glioxilatos/metabolismo , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/metabolismo , Oxidación-Reducción , Espectrofotometría/métodos , Factores de Tiempo

RESUMEN

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1alpha (45.5 kDa) and two E1beta (37.5 kDa) subunits forming an alpha2 beta2 tetramer. In patients with type IA maple syrup urine disease, the E1alpha subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1alpha missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1alpha and untagged E1beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-alpha), moderately slow (G245R-alpha), slow (G204S-alpha, A240P-alpha, F364C-alpha, Y368C-alpha, and Y393N-alpha), and no (T265R-alpha) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 degreesC (in the case of T265R-alpha), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as alpha2 beta2 tetramers. In contrast, a subset of E1alpha missense mutations caused the occurrence of exclusive alphabeta dimers (Y393N-alpha and F364C-alpha) or of both alpha2beta2 tetramers and lower molecular weight species (Y368C-alpha and T265R-alpha). Thermal denaturation at 50 degreesC indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min-1), with the T265R-alpha mutant E1 most severely affected (rate constant, 4.45 min-1). The results establish that the human E1alpha mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha, have no effect on E1 subunit assembly. The T265R-alpha mutation adversely impacts both E1alpha folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native alpha2 beta2 structure.

Asunto(s)

Cetona Oxidorreductasas/metabolismo , Enfermedad de la Orina de Jarabe de Arce/enzimología , Complejos Multienzimáticos/metabolismo , 3-Metil-2-Oxobutanoato Deshidrogenasa (Lipoamida) , Secuencia de Aminoácidos , Animales , Catálisis , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Electroforesis en Gel de Campo Pulsado , Humanos , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/aislamiento & purificación , Cinética , Enfermedad de la Orina de Jarabe de Arce/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Mutación , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido

RESUMEN

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953-3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por, oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. Both porD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB and oorA appeared to be lethal.

Asunto(s)

Helicobacter pylori/genética , Cetona Oxidorreductasas/genética , NADP/metabolismo , Secuencia de Aminoácidos , Archaea/enzimología , Archaea/genética , Sitios de Unión , Cationes/metabolismo , Clonación Molecular , Transporte de Electrón , Escherichia coli/genética , Flavodoxina/genética , Flavodoxina/metabolismo , Genes Bacterianos , Genoma Bacteriano , Helicobacter/enzimología , Helicobacter/genética , Helicobacter pylori/enzimología , Helicobacter pylori/metabolismo , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Cetona Oxidorreductasas/metabolismo , Datos de Secuencia Molecular , NAD/metabolismo , Sistemas de Lectura Abierta , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Nucleótidos de Timina/metabolismo , Transformación Bacteriana