RESUMO
Bartonella spp. are globally distributed bacteria that cause endocarditis in humans and domestic animals. Recent work has suggested bats as zoonotic reservoirs of some human Bartonella infections; however, the ecological and spatiotemporal patterns of infection in bats remain largely unknown. Here we studied the genetic diversity, prevalence of infection across seasons and years, individual risk factors, and possible transmission routes of Bartonella in populations of common vampire bats (Desmodus rotundus) in Peru and Belize, for which high infection prevalence has previously been reported. Phylogenetic analysis of the gltA gene for a subset of PCR-positive blood samples revealed sequences that were related to Bartonella described from vampire bats from Mexico, other Neotropical bat species, and streblid bat flies. Sequences associated with vampire bats clustered significantly by country but commonly spanned Central and South America, implying limited spatial structure. Stable and nonzero Bartonella prevalence between years supported endemic transmission in all sites. The odds of Bartonella infection for individual bats was unrelated to the intensity of bat flies ectoparasitism, but nearly all infected bats were infested, which precluded conclusive assessment of support for vector-borne transmission. While metagenomic sequencing found no strong evidence of Bartonella DNA in pooled bat saliva and fecal samples, we detected PCR positivity in individual saliva and feces, suggesting the potential for bacterial transmission through both direct contact (i.e., biting) and environmental (i.e., fecal) exposures. Further investigating the relative contributions of direct contact, environmental, and vector-borne transmission for bat Bartonella is an important next step to predict infection dynamics within bats and the risks of human and livestock exposures.
Assuntos
Infecções por Bartonella/veterinária , Bartonella/classificação , Bartonella/genética , Quirópteros/microbiologia , Transmissão de Doença Infecciosa , Variação Genética , Animais , Proteínas de Bactérias/genética , Bartonella/isolamento & purificação , Infecções por Bartonella/epidemiologia , Infecções por Bartonella/transmissão , Belize , Sangue/microbiologia , Análise por Conglomerados , Fezes/microbiologia , Glutamato Sintase/genética , Peru , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Fatores de Risco , Saliva/microbiologia , Estações do Ano , Análise de Sequência de DNARESUMO
Four Amblyomma sabanerae ticks collected from a turtle (Kinosternon sp.) in San Miguel, El Salvador, were found by molecular analysis to be infected by Rickettsia bellii. We provide the first report of Rickettsia bellii in Central America, and the first report of a Rickettsia species in El Salvador.
Assuntos
Ixodidae/microbiologia , Rickettsia/classificação , Rickettsia/isolamento & purificação , Animais , DNA Bacteriano/química , DNA Bacteriano/genética , El Salvador , Glutamato Sintase/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Rickettsia/genética , Análise de Sequência de DNA , Tartarugas/parasitologiaAssuntos
Bartonella quintana/isolamento & purificação , Infestações por Piolhos/parasitologia , Pediculus/microbiologia , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Glutamato Sintase/genética , Humanos , México , Reação em Cadeia da Polimerase , Prevalência , Rickettsia prowazekii/isolamento & purificaçãoRESUMO
An immunohistochemical analysis of brain subcortical white matter astroglia from human (infant, adult) and adult monkey (Cebus apella, Macaca nemestrina) cases without any known neurological disease, is described. Expression of synaptic vesicle-associated proteins, excitatory amino acid transporters (EAAT1 and EAAT2) and GABAA Ralpha2 receptor produced coarse punctate labeling in human adult white matter astrocytes. A finer, generalized, punctate labeling was observed in human infants and adult C. apella monkeys. Labeling of neuronal somata and processes with microtubule-associated proteins (MAP2a-c) and neuron nuclear (NeuN) antibodies, was also observed in subcortical white matter of humans and monkeys. Results suggest competence of subcortical white matter astroglia of the primate brain to participate in various transmitter regulatory pathways. It is also proposed that, collectively with resident neurons, they may exert some role in affecting the transfer of information that takes place through the various associational and projecting fiber systems coursing through this brain compartment.
Assuntos
Astrócitos/metabolismo , Encéfalo/citologia , Neurônios/fisiologia , Primatas/fisiologia , Idoso , Animais , Biomarcadores , Cebus , Conexina 43 , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato Sintase/metabolismo , Humanos , Imuno-Histoquímica , Macaca fascicularis , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/fisiologia , Neuropeptídeo Y/metabolismo , Neurotransmissores/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Sinaptotagminas/metabolismoRESUMO
The role of glutamate as osmoprotector was investigated through the study of a mutation in its biosynthetic pathway. A glt::Tn917-lacZ-cat insertion mutant (N1) conferring glutamate auxotrophy and enhanced beta-galactosidase expression on high-salt media was selected. Co-transformation experiments and PCR analysis allowed locating the insertion into the gltB gene corresponding to the small unit of the glutamate synthase (GOGAT). The N1 mutant strain presented a glutamate requirement for growth and a tenfold decrease in GOGAT activity. Transcriptional activity of GOGAT, measured as beta-galactosidase from the transposon fusion, correlated with enzymatic activity; expression was enhanced at the stationary phase and in high-ionic-strength media. However, osmotolerance of cultures of N1 mutant were as wild-type (wt), at least in semi-rich medium. In contrast, sporulation was slightly reduced (75% of wt), and spores were less resistant to UV, heat, and osmolarity, properties linked to the content of small, acid-soluble proteins (SASP). The content of these proteins was, in fact, reduced, in particular the SASP-gamma type. The peptidoglycan-cortex, however, was not impaired since spores maintained lysozyme resistance. Addition of glutamate during sporulation partially rescued spore resistance, but germination and outgrowth remained impaired. Deficiencies in germination and outgrowth were also observed with spores from a gltA mutant strain. Taken together, these results pointed to the importance of GOGAT activity during sporulation, in particular for the synthesis SASPs.
Assuntos
Bacillus subtilis/fisiologia , Glutamato Sintase/genética , Glutamato Sintase/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Fusão Gênica Artificial , Bacillus subtilis/enzimologia , Proteínas de Bactérias/biossíntese , Elementos de DNA Transponíveis , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reporter , Ácido Glutâmico/biossíntese , Muramidase/metabolismo , Mutagênese Insercional , Pressão Osmótica , Peptidoglicano/biossíntese , Transcrição Gênica , beta-Galactosidase/metabolismoRESUMO
Azospirillum brasilense glutamate synthase (GltS) is the prototype of bacterial NADPH-dependent enzymes, a class of complex iron-sulfur flavoproteins essential in ammonia assimilation processes. The catalytically active GltS alpha beta holoenzyme and its isolated alpha and beta subunits (162 and 52 kDa, respectively) were analyzed using synchrotron radiation x-ray solution scattering. The GltS alpha subunit and alpha beta holoenzyme were found to be tetrameric in solution, whereas the beta subunit was a mixture of monomers and dimers. Ab initio low resolution shapes restored from the scattering data suggested that the arrangement of alpha subunits in the (alpha beta)4 holoenzyme is similar to that in the tetrameric alpha 4 complex and that beta subunits occupy the periphery of the holoenzyme. The structure of alpha 4 was further modeled using the available crystallographic coordinates of the monomeric alpha subunit assuming P222 symmetry. To model the entire alpha beta holoenzyme, a putative alpha beta protomer was constructed from the coordinates of the alpha subunit and those of the N-terminal region of porcine dihydropyrimidine dehydrogenase, which is similar to the beta subunit. Rigid body refinement yielded a model of GltS with an arrangement of alpha subunits similar to that in alpha 4, but displaying contacts also between beta subunits belonging to adjacent protomers. The holoenzyme model allows for independent catalytic activity of the alpha beta protomers, which is consistent with the available biochemical evidence.
Assuntos
Azospirillum brasilense/enzimologia , Glutamato Sintase/química , NADP/química , Animais , Catálise , Cristalografia por Raios X , Di-Hidrouracila Desidrogenase (NADP) , Dimerização , Modelos Biológicos , Modelos Moleculares , Oxirredutases/química , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espalhamento de Radiação , Suínos , Síncrotrons , Raios XRESUMO
A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8% of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Biblioteca Gênica , Glutamato Sintase/genética , Miosinas/genética , Plasmodium falciparum/genética , AnimaisRESUMO
A cDNA library of Plasmodium falciparum (Colombian strain FCB2) asexual stage was constructed in the lambda ZipLox vector. The lambda ZipLox library and a lambda ZAPII (Dd2 strain) were screened for genes coding for proteins that bind with or are related to calmodulin (CaM). Screening was accomplished with Hot start PCR assays and hybridization with radiolabeled probes. Actin I, CaM, glutamate synthase (GOGAT) and the three myosin clones--Pfmyo A, Pfmyo B and Pfmyo C--were identified. The clones coding for actin I, CaM and GOGAT were retrieved from the lambda ZipLox library, and the GOGAT and Pfmyo A clones from the lambda ZAP II library. The GOGAT clone contained an insert of 2,413 base pairs corresponding to 24.8 of the reported sequence. The Pfmyo A insert was 2,457 base pairs long, and represented the complete mRNA coding for this gene. Finally, the first report of a complete cDNA clone containing the P. falciparum myosin A is presented.
Assuntos
Animais , Proteínas de Ligação a Calmodulina , Biblioteca Gênica , Glutamato Sintase , Miosinas , Plasmodium falciparumRESUMO
The effect of aluminum on the activity of PLC was examined in transformed roots from Catharanthus roseus (L) G. Don. When added in vitro to the reaction mixture, Al inhibited the enzymatic activity in a concentration and time-dependent fashion. This effect is very similar for both activities (soluble and membrane-associated). When roots were treated in vivo with Al 0.1 mM for short periods (0-4 h), PLC activity was also inhibited. Aluminum (1 mM) diminished root growth in approximately 50% when added on the first day of the culture cycle conditions in which PLC activity is also affected. Other enzymatic activities (NAD+-GDH, NADH-GDH, NADH-GOGAT and HMGR) were not affected when roots were treated with Al (0.1 mM) for short periods of time (1 h). Results obtained in this work suggest that the Al can affect PLC activity as a specific target. Enzymes: Phospholipase C (EC 3.1.4.10).
Assuntos
Alumínio/farmacologia , Magnoliopsida/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Glutamato Desidrogenase/metabolismo , Glutamato Sintase/metabolismo , Concentração de Íons de Hidrogênio , Hidroximetilglutaril-CoA Redutases/metabolismo , Magnoliopsida/enzimologia , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Azospirillum brasilense glutamate synthase is a complex iron-sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield L-glutamate. Its catalytically active alphabeta protomer is composed of two dissimilar subunits (alpha subunit, 164.2 kDa; beta subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the beta subunit), one FMN (at Site 2, the 2-oxoglutarate/L-glutamate site in the alpha subunit) and three different iron-sulfur clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the glutamate synthase beta and alpha subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense glutamate synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2',5' ADP-Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of glutamate synthase to form a stable (alphabeta)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated beta subunit does not oligomerize, while the isolated glutamate synthase alpha subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2'-phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that glutamate synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the alpha subunit. At the same time, the strict requirement of a 2'-phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP+ analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP+, which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.
Assuntos
Azospirillum brasilense/enzimologia , Glutamato Sintase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Nucleotídeos de Adenina/farmacologia , Catálise , Plasmídeos , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
Saccharomyces cerevisiae glutamate synthase (GOGAT) is an oligomeric enzyme composed of three 199-kDa identical subunits encoded by GLT1. In this work, we analyzed GLT1 transcriptional regulation. GLT1-lacZ fusions were prepared and GLT1 expression was determined in a GDH1 wild-type strain and in a gdh1 mutant derivative grown in the presence of various nitrogen sources. Null mutants impaired in GCN4, GLN3, GAT1/NIL1, or UGA43/DAL80 were transformed with a GLT1-lacZ fusion to determine whether the above-mentioned transcriptional factors had a role in GLT1 expression. A collection of increasingly larger 5' deletion derivatives of the GLT1 promoter was constructed to identify DNA sequences that could be involved in GLT1 transcriptional regulation. The effect of the lack of GCN4, GLN3, or GAT1/NIL1 was also tested in the pertinent 5' deletion derivatives. Our results indicate that (i) GLT1 expression is negatively modulated by glutamate-mediated repression and positively regulated by Gln3p- and Gcn4p-dependent transcriptional activation; (ii) two cis-acting elements, a CGGN15CCG palindrome and an imperfect poly(dA-dT), are present and could play a role in GLT1 transcriptional activation; and (iii) GLT1 expression is moderately regulated by GCN4 under amino acid deprivation. Our results suggest that in a wild-type strain grown on ammonium, GOGAT constitutes an ancillary pathway for glutamate biosynthesis.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Glutamato Sintase/genética , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Glutamato Sintase/química , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.
Assuntos
Glutamato Desidrogenase/genética , Glutamato Sintase/metabolismo , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/biossíntese , Kluyveromyces/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Genes Bacterianos , Kluyveromyces/enzimologia , Kluyveromyces/genética , Dados de Sequência Molecular , MutaçãoRESUMO
The pH dependence of the kinetic parameters of the glutamine- and ammonia-dependent reactions of Azospirillum brasilense glutamate synthase revealed the presence of ionizable groups with pKa values between 6 and 10 involved in the binding of the substrates and in catalytic steps. The V profile of the glutamine-dependent reaction is complicated by a deviation from a simple bell-shaped curve between pH 8 and pH 10, which may suggest that deprotonation of a group with pKa value in this region decreases but does not abolish glutamine-dependent enzyme activity. This group does not seem to be required in the ammonia-dependent reaction of GltS, which decreases on the acidic and alkaline sides as groups with pKa values of about 8.8 and 9.9 dissociate. The V/K profile for ammonia exhibits a single pKa value of about 8.7, suggesting that ammonia is the actual substrate of the enzyme, and that ammonia binding to glutamate synthase is largely pH independent. The hypothesis that a group with pKa between 8 and 10 is involved in the glutaminase segment of the glutamine-dependent glutamate synthase activity was supported by studies of the modification of the enzyme by 6-diazo-5-oxo-L-norleucine, a glutamine analog, and iodoacetamide, a cysteine-directed reagent. Analyses of the kinetics of inactivation of the enzyme in the presence and absence of enzyme substrates and their analogs at different pH values demonstrated that iodoacetamide reacts with a group involved in glutamine binding and/or activation, most likely the cysteine residue at the N-terminus of glutamate synthase alpha subunit, which may form a Cys-His ion pair in the active site of glutamate synthase, as suggested for other amidotransferases (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619).
Assuntos
Azospirillum brasilense/enzimologia , Cisteína/química , Glutamato Sintase/metabolismo , Histidina/química , Iodoacetamida/farmacologia , Sequência de Aminoácidos , Amônia/farmacologia , Catálise , Glutamato Sintase/antagonistas & inibidores , Glutamato Sintase/química , Glutamina/metabolismo , Glutamina/farmacologia , Concentração de Íons de Hidrogênio , Iodoacetamida/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Cinética , Dados de Sequência Molecular , NADP/metabolismo , NADP/farmacologia , Análise de SequênciaRESUMO
An Azospirillum brasilense mutant (N12) pleiotropically defective in the assimilation of nitrogenous compounds (Asm-) was isolated and found lacking in the glutamate synthase (GOGAT-). The glt (GOGAT) locus of A. brasilense was identified by isolating a broad-host-range pLAFR1 cosmid clone from a gene library of the bacterium that rectified Asm- and GOGAT- defects (full recovery of activities of the nitrogenase, the assimilatory nitrate and nitrite reductases, and the glutamate synthase). A 7.5-kb EcoRI fragment of the cosmid clone that also complemented N12 was partially sequenced to identify the open reading frame for the alpha-subunit of GOGAT. The amino acid sequences deduced from the partial nucleotide sequences of the glt locus of A. brasilense showed considerable homology with that of the alpha-subunit of GOGAT coded by the gltB gene of Escherichia coli. The genetic lesion of N12 was found within the gltB gene of A. brasilense. The gltB promoter of A. brasilense showed the presence of a consensus sigma-70-like recognition site (as in E. coli) in addition to potential NtrA-RNA polymerase, IHF, and NifA binding sites.
Assuntos
Azospirillum brasilense/genética , Genes Bacterianos , Glutamato Sintase/genética , Mutação , Sequência de Aminoácidos , Cloreto de Amônio/metabolismo , Azospirillum brasilense/enzimologia , Azospirillum brasilense/metabolismo , Sequência de Bases , Clonagem Molecular , Cosmídeos , Escherichia coli/genética , Biblioteca Gênica , Glutamatos/metabolismo , Ácido Glutâmico , Glutamina/metabolismo , Dados de Sequência Molecular , Nitrogenase/metabolismo , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli. Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A. brasilense GltS. The organization of the gltBD region of A. brasilense differs from that of the corresponding region in E. coli: in A. brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB. The deduced amino acid sequences reveal a high similarity with GltS from E. coli and with the ferredoxin-dependent GltS from maize. Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins.
Assuntos
Azospirillum brasilense/enzimologia , Azospirillum brasilense/genética , Genes Bacterianos , Glutamato Sintase/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Óperon , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.
Assuntos
Azospirillum brasilense/enzimologia , Flavinas/química , Glutamato Sintase/química , Proteínas Ferro-Enxofre/química , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Ferro/química , Luz , NADP/química , Oxirredução , Análise Espectral , Sulfitos/química , Enxofre/química , TemperaturaRESUMO
A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.
Assuntos
Escherichia coli/genética , Glutamato Sintase/genética , Glutamatos/metabolismo , Saccharomyces cerevisiae/genética , Northern Blotting , Clonagem Molecular , Escherichia coli/enzimologia , Genes Fúngicos , Teste de Complementação Genética , Glutamato Sintase/metabolismo , Ácido Glutâmico , Cinética , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Temperatura , Transformação GenéticaRESUMO
The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.
Assuntos
Azospirillum brasilense/enzimologia , Glutamato Sintase/metabolismo , Amônia/metabolismo , Glutamatos/biossíntese , Ácido Glutâmico , Glutaminase/metabolismo , Glutamina/metabolismo , Glutamina/farmacologia , Ácidos Cetoglutáricos/metabolismo , Espectroscopia de Ressonância Magnética , NADP/metabolismo , Oxirredução , Piridinas/metabolismoRESUMO
The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.
Assuntos
Azospirillum brasilense/enzimologia , Glutamato Sintase/metabolismo , Amônia/farmacologia , Glutamato Sintase/antagonistas & inibidores , Glutamina/metabolismo , Glutamina/farmacologia , Ácidos Cetoglutáricos/metabolismo , Cinética , NADP/metabolismo , Oxirredução , Espectrofotometria , Especificidade por Substrato , TrítioRESUMO
The amino acid composition and the N-terminal sequences of the two dissimilar subunits of glutamate synthase from Azospirillum brasilense have been determined along with the sequences of selected CNBr peptides. Comparison of our data with those available for Escherichia coli glutamate synthase revealed an overall good homology between the enzymes from the two sources. This is more evident for the heavy subunits where the highly conserved N-terminal sequence containing Cys-1, suggests that this region may be involved in catalysis. However, it appears that the light subunits are different with respect to both their amino acid composition and their N-terminal region, suggesting that the latter may not be part of the enzyme active site. Finally, an extinction coefficient at 444 nm of 62.66 +/- 4.61 mM-1.cm-1 was determined.