RESUMO
The Starmerella bombicola lactone esterase (SBLE) is a novel enzyme that, in vivo, catalyzes the intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment. In fact, this is an unusual reaction given the unfavorable conditions for dehydration. This characteristic strongly contributes to the potential of SBLE to become a 'green' tool in industrial applications. Indeed, lactonization occurs normally in organic solvents, an application for which microbial lipases are increasingly used as biocatalysts. Previously, we described the production of recombinant SBLE (rSBLE) in Pichia pastoris (syn. Komagataella phaffii). However, expression was not optimal to delve deeper into the enzyme's potential for industrial application. In the current study, we explored codon-optimization of the SBLE gene and we optimized the rSBLE expression protocol. Temperature reduction had the biggest impact followed by codon-optimization and co-expression of the HAC1 transcription factor. Combining these approaches, we achieved a 32-fold improvement of the yield during rSBLE production (from 0.75 mg/l to 24 mg/L culture) accompanied with a strong reduction of contaminants after affinity purification.
Assuntos
Esterases/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomycetales/enzimologia , Códon/genética , Esterases/química , Esterases/genética , Esterases/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Química Verde , Lactonas/metabolismo , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomycetales/genética , TemperaturaRESUMO
Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production.
Assuntos
Regulação da Expressão Gênica/fisiologia , Pichia/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma/metabolismo , Animais , Doenças do Cão/prevenção & controle , Cães , Feminino , Proteínas de Protozoários/genética , Fatores de Tempo , Trypanosoma/isolamento & purificaçãoRESUMO
Chemotherapy remains mainly used for the treatment of acute myeloid leukemia (AML). However, in the past 3 decades limited progress has been achieved in improving the long-term disease-free survival. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin core targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine moiety facilitates F14512 selective uptake by tumour cells via the polyamine transport system, a machinery overactivated in cancer cells. F14512 has been characterized as a potent drug candidate and is currently in Phase I clinical trials. Here, we demonstrated marked survival benefit and therapeutic efficacy of F14512 treatments in a series of human AML models, established either from AML cell lines or from patient AML samples. Furthermore, we reported in vitro synergistic anti-leukemic effects of F14512 in combination with cytosine arabinoside (Ara-C), doxorubicin, gemcitabine, bortezomib or SAHA. In vivo combination of suboptimal doses of F14512 with Ara-C also resulted in enhanced anti-leukemic activity. We further showed that F14512 triggered both senescence and apoptosis in vivo in primary AML models, but not autophagy. Overall, these results support the clinical development in onco-hematology of this novel promising drug candidate.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sangue Fetal/efeitos dos fármacos , Subunidade gama Comum de Receptores de Interleucina/fisiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Animais , Western Blotting , Ácidos Borônicos/administração & dosagem , Bortezomib , Citarabina/administração & dosagem , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Doxorrubicina/administração & dosagem , Sangue Fetal/citologia , Citometria de Fluxo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Podofilotoxina/administração & dosagem , Podofilotoxina/análogos & derivados , Pirazinas/administração & dosagem , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Vorinostat , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1-9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC(50) values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.
Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Proteassoma , Inibidores da Topoisomerase II , Acridinas/síntese química , Acridinas/química , Acridinas/classificação , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/classificação , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/classificação , Células HL-60 , Humanos , Concentração Inibidora 50 , Leucemia Promielocítica Aguda/tratamento farmacológico , Modelos Biológicos , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/análise , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2. For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2. We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2. We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Proteobactérias/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte de Elétrons , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Estrutura Molecular , Oxirredução , Filogenia , Conformação Proteica , Proteobactérias/classificação , Proteobactérias/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.
Assuntos
Ectothiorhodospira/enzimologia , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Sequência de Aminoácidos , Proteínas de Bactérias , Carbamatos/química , Espectrometria de Massas , Peptídeos/síntese química , Peptídeos/químicaRESUMO
The crystal structure of the major endoglucanase from the thermophilic fungus Thermoascus aurantiacus was determined by single isomorphous replacement at 1.12A resolution. The full sequence supports the classification of the protein in a subgroup of glycoside hydrolase family 5 for which no structural data are available yet. The active site shows eight critical residues, strictly conserved within family 5. In addition, aromatic residues that line the substrate-binding cleft and that are possibly involved in substrate-binding are identified. A number of residues seem to be conserved among members of the subtype, including a disulphide bridge between Cys212 and Cys249.
Assuntos
Ascomicetos/enzimologia , Celulase/química , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de AminoácidosRESUMO
Pseudomonas putida contains an amine dehydrogenase that is called a quinohemoprotein as it contains a quinone and two hemes c as redox active groups. Amino acid sequence analysis of the smallest (8.5 kDa), quinone-cofactor-bearing subunit of this heterotrimeric enzyme encountered difficulties in the interpretation of the results at several sites of the polypeptide chain. As this suggested posttranslational modifications of the subunit, the structural genes for this enzyme were determined and mass spectrometric de novo sequencing was applied to several peptides obtained by chemical or enzymatic cleavage. In agreement with the interpretation of the X-ray electronic densities in the diffraction data for the holoenzyme, our results show that the polypeptide of the small subunit contains four intrachain cross-linkages in which the sulfur atom of a cysteine residue is involved. Two of these cross-linkages occur with the beta-carbon atom of an aspartic acid, one with the gamma-carbon atom of a glutamic acid and the fourth with a tryptophanquinone residue, this adduct constituting the enzyme's quinone cofactor, CTQ. The thioether type bond in all four of these adducts has never been found in other proteins. CTQ is a novel cofactor in the series of the recently discovered quinone cofactors.
Assuntos
Cisteína/química , Indolquinonas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Pseudomonas putida/enzimologia , Sulfetos/química , Triptofano/análogos & derivados , Sequência de Aminoácidos , Aminoácidos/química , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , Ácido Glutâmico/química , Heme/química , Espectrometria de Massas , Modelos Químicos , Modelos Genéticos , Dados de Sequência Molecular , Fases de Leitura Aberta , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Peptídeos/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Quinonas/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Triptofano/química , Raios XRESUMO
Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.
Assuntos
Grupo dos Citocromos c , Oxirredutases , Shewanella/enzimologia , Sequência de Aminoácidos , Anaerobiose , Grupo dos Citocromos c/química , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Fumaratos/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Oxirredução , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Análise de Sequência de DNA , Shewanella/crescimento & desenvolvimento , Transcrição GênicaRESUMO
Carp beta-endorphin is posttranslationally modified by N-terminal acetylation and C-terminal cleavage. These processes determine the biological activity of the beta-endorphins. Forms of beta-endorphin were identified in the pars intermedia and the pars distalis of the pituitary gland of the common carp (Cyprinus carpio), as well as the forms released in vitro and into the blood. After separation and quantitation by high performance liquid chromatography (HPLC) coupled with radioimmunoassay, the beta-endorphin immunoreactive products were identified by electrospray ionisation mass spectrometry and peptide sequencing. The release of beta-endorphins by the pituitary gland was studied after stimulation with corticotrophin-releasing factor (CRF) in vitro. In the pars intermedia, eight N-acetylated truncated forms were identified. Full length N-acetyl beta-endorphin(1-33) coeluted with N-acetyl beta-endorphin(1-29) and these forms together amounted to over 50% of total immunoreactivity. These products were partially processed to N-acetyl betaendorphin(1-15) (30.8% of total immunoreactivity) and N-acetyl beta-endorphin(1-10) (3.1%) via two different cleavage pathways. The acetylated carp homologues of mammalian alpha- and gamma-endorphin were also found. N-acetyl beta-endorphin(1-15) and (1-29) and/or (1-33) were the major products to be released in vitro, and were the only acetylated beta-endorphins found in blood plasma, although never together. CRF stimulated the release of opioid beta-endorphin from the pars distalis. This non-acetylated beta-endorphin represents the full length peptide and is the most abundant form in plasma.
Assuntos
Carpas/metabolismo , Hipófise/química , beta-Endorfina/análogos & derivados , beta-Endorfina/análise , Animais , Cromatografia Líquida de Alta Pressão , Hormônio Liberador da Corticotropina/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Peso Molecular , Hipófise/anatomia & histologia , Hipófise/metabolismo , Radioimunoensaio , Espectrometria de Massas por Ionização por Electrospray , Estimulação Química , beta-Endorfina/sangueRESUMO
New polypeptide components have been isolated from Dendroaspis angusticeps venom using chromatography. Two polypeptides containing 59 and 57 amino acids, called 'DaE1' and 'DaE2' respectively, have been purified to homogeneity and fully sequenced. Spectrometric analysis yielded masses of 6631.5 and 6389.0 Da, respectively. The polypeptides share 98 and 95% identity, respectively, with trypsin inhibitor E (DpE) of Dendroaspis polylepis polylepis. 'DaE' polypeptides inhibit Kv1.1 channels with an IC(50) value in the range of 300 nM. They can be considered as new dendrotoxins, albeit with fairly low affinity as compared to alpha-DTX. 'DaE' polypeptides do not affect Kir2.1 channels.
Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/isolamento & purificação , Elapidae , Peptídeos/química , Peptídeos/isolamento & purificação , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Células Cultivadas , Cromatografia em Gel , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Venenos Elapídicos/farmacologia , Canal de Potássio Kv1.1 , Dados de Sequência Molecular , Peso Molecular , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio , Canais de Potássio/genética , Canais de Potássio/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Xenopus laevisRESUMO
Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586-R602 (SR-17) and is phosphorylated at one or two serine residues. Another novel peptide H603-Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.
Assuntos
Grânulos Cromafim/química , Cromograninas/química , Fragmentos de Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia de Afinidade , Cromogranina B , DNA Complementar/genética , Cromatografia Gasosa-Espectrometria de Massas/métodos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , SuínosRESUMO
An extracellular beta-glucosidase from Thermoascus aurantiacus was purified to homogeneity by DEAE-Sepharose, Ultrogel AcA 44 and Mono-P column chromatography. The enzyme was a homotrimer, with a monomer molecular mass of 120 kDa; only the trimer was optimally active at 80 degrees C and at pH 4.5. At 90 degrees C, the enzyme showed 70% of its optimal activity. It was stable at pH 5.2 and at temperatures up to 70 degrees C for 48 h, but stability decreased above 70 degrees C and at pH values above and below 5.0. The enzyme hydrolysed aryl and alkyl beta-d-glucosides and cello-oligosaccharides, and was specific for substrates with a beta-glycosidic linkage. The hydroxy groups at positions 2, 4 and 6 of a glucose residue at the non-reducing end of a disaccharide appeared to be essential for catalysis. The enzyme had the lowest K(m) towards p-nitrophenyl beta-d-glucoside (0.1137 mM) and the highest k(cat) towards cellobiose and beta,beta-trehalose (17052 min(-1)). It released one glucose unit at a time from the non-reducing end of cello-oligosaccharides, and the rate of hydrolysis decreased with an increase in chain length. Glucose and d-delta-gluconolactone inhibited the beta-glucosidase competitively, with K(i) values of 0.29 mM and 8.3 nM respectively, while methanol, ethanol and propan-2-ol activated the enzyme. The enzyme catalysed the synthesis of methyl, ethyl and propyl beta-d-glucosides in the presence of methanol, ethanol and propan-2-ol respectively with either glucose or cellobiose, although cellobiose was preferred. An acidic pH favoured hydrolysis and transglycosylation, but high concentrations of alcohols favoured the latter reaction. The stereochemistry of cellobiose hydrolysis revealed that beta-glucosidase from T. aurantiacus is a retaining glycosidase, while N-terminal amino acid sequence alignment indicated that it is a member of glycoside hydrolase family 3.
Assuntos
Ascomicetos/enzimologia , beta-Glucosidase/química , beta-Glucosidase/metabolismo , Álcoois/farmacologia , Sequência de Aminoácidos , Celobiose/metabolismo , Cromatografia Líquida de Alta Pressão , Dissacarídeos/metabolismo , Estabilidade Enzimática , Gluconatos/farmacologia , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Cinética , Lactonas , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Polissacarídeos/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Especificidade por Substrato , Temperatura , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/isolamento & purificaçãoRESUMO
The complete sequence of Bacillus pasteurii cytochrome c-553 was determined by standard methods of Edman degradation of overlapping peptides combined with mass spectrometry. The protein contains 92 residues and a single heme-binding site. It is most similar to Bacillus licheniformis, Bacillus PS3, and Bacillus subtilis cytochromes c-551, which are lipoproteins that are partially solubilized through proteolytic cleavage of the N-terminal diacyl-glyceryl-cysteine membrane anchor. The high yield of the B. pasteurii cytochrome c-553, together with evidence that shorter forms of the cytochrome occur in the mixture of otherwise pure protein, suggests that the membrane anchor is very susceptible to proteolysis and that the soluble form of the cytochrome is therefore released from the membrane upon cell breakage. A sequence-based calculation of the protein secondary structure suggests the presence of a typical cytochrome helical fold with a random-coil N-terminus tail.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Lipoproteínas/química , Sequência de Aminoácidos , Sítios de Ligação , Grupo dos Citocromos c/isolamento & purificação , Espectrometria de Massas , Metaloendopeptidases , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Estrutura Secundária de Proteína , Alinhamento de Sequência , SolubilidadeRESUMO
Cytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 microM and allowing a maximal catalytic centre activity of 116,000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature.
Assuntos
Proteínas de Bactérias/química , Citocromo-c Peroxidase/química , Pseudomonas/enzimologia , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas de Bactérias/isolamento & purificação , Grupo dos Citocromos c/química , Citocromo-c Peroxidase/genética , Citocromo-c Peroxidase/isolamento & purificação , Ativação Enzimática , Expressão Gênica , Dados de Sequência Molecular , Peso Molecular , Concentração Osmolar , Oxirredução , Periplasma/enzimologia , Pseudomonas/genética , Alinhamento de Sequência , Espectrofotometria UltravioletaRESUMO
Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.
Assuntos
Caseínas/metabolismo , Endopeptidases/metabolismo , Lactobacillus/enzimologia , Sequência de Aminoácidos , Caseínas/química , Parede Celular/enzimologia , Queijo/microbiologia , Endopeptidases/isolamento & purificação , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The complete amino acid sequence of the low-redox potential cytochrome c-551.5 from Rhodobacter sphaeroides was determined by automated Edman degradation combined with mass spectroscopy. There are 139 residues and two typical Cys-X-X-Cys-His heme-binding sites. A homologous low-redox potential cytochrome was also sequenced from Rhodobacter adriaticus and was found to contain 126 residues. It is 53% identical to that of Rb. sphaeroides and has two internal deletions of one and five residues. The Rhodobacter diheme cytochromes are 21-24% identical to the translated open reading frame SLL1886 from Synechocystis sp. PCC6801. There are at least two deletions of five and eight residues in the 188-residue cyanobacterial protein. Each of the three cytochromes has more histidines than it needs to bind the two hemes, but conserved histidines located 23 residues after the first heme and 14-19 residues before the second heme are likely to be the sixth heme ligands. There is no evidence for gene doubling and no similarity to any other known cytochromes. The measured helix content of 24% is much less than normal for c-type cytochromes. These proteins thus appear to be representative of an entirely new class of c-type cytochromes.
Assuntos
Proteínas de Bactérias , Grupo dos Citocromos c/química , Heme/química , Rhodobacter sphaeroides/enzimologia , Rhodobacter/enzimologia , Sequência de Aminoácidos , Grupo dos Citocromos c/isolamento & purificação , Grupo dos Citocromos c/metabolismo , Heme/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The complete amino acid sequence of the blue copper-containing nitrite reductase enzyme (NiR) from Achromobacter xylosoxidans has been determined by chemical analysis, supported by high precision mass analysis. The polypeptide chain contains 336 residues with an overall charge of 0, including the +2 state of each of the copper ions. The two NiR enzymes for which the three-dimensional structures have been solved are green in color and have different absorption spectra than those of the blue-colored protein from A. xylosoxidans. The ligands to the two copper atoms are conserved. Therefore, the difference between the blue and the green NiR must depend on subtle changes in the geometry of the type I copper-sulfur bond. Both overall protein charge and active site charge are different in A. xylosoxidans NiR which may reflect the use of azurin as electron donor as opposed to the other enzymes that use pseudoazurin.
Assuntos
Alcaligenes/enzimologia , Cobre/química , Nitrito Redutases/química , Sequência de Aminoácidos , Azurina/metabolismo , Sítios de Ligação/fisiologia , Transporte de Elétrons/fisiologia , Endopeptidases/metabolismo , Espectrometria de Massas , Metaloproteínas/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Alinhamento de Sequência , Análise de SequênciaRESUMO
The amino acid sequence of cytochrome c peroxidase from Pseudomonas aeruginosa has been determined using classical chemical degradation techniques combined with accurate mass analysis of all the generated peptides. The sequence obtained is composed of 346 amino acids and confirms the recently published cDNA-derived sequence except at one position [Ridout et al. (1995) FEBS Lett. 365, 152-154]. Based on this sequence, we propose a new model for the binding of the peroxide and the cytochrome electron donor to CCP which is in essence the reverse of the one proposed by Ellfolk et al.