RESUMO
A new purification procedure for the multienzyme of gramicidin S-synthetase has been developed. In vitro proteolysis with partial inactivation is suppressed by protease inhibitors EDTA, phenylmethylsulfonylfluoride, and fast preparation methods during initial separation steps. Activity has only been assayed by the total reaction of gramicidin S-synthetase, not by partial reactions of amino acid activation. The assay has been improved by evaluation of inhibitory concentrations of buffers, salts, and the product gramicidin S. It has been demonstrated that the rate of peptide synthesis in extracts containing both enzymes of gramicidin S-synthetase depends on protein concentration in a second order function. The multienzyme or heavy enzyme has been purified about 1400-fold to a specific activity of 24 nM/min per mg of protein, and the relation of this activity to the calculated in vivo activity is discussed.
Assuntos
Isomerases de Aminoácido/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Peptídeo Sintases/isolamento & purificação , Bacillus/enzimologia , Ácido Edético/farmacologia , Gramicidina/biossíntese , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Peso Molecular , Ornitina/metabolismo , Concentração Osmolar , Fosfatos/farmacologiaRESUMO
1. A method characterizing the fully active gramicidin S-synthetase (EC. 6.3.2.-) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. The molecular weight of 280000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300000 daltons has been determined, correlating either mobilities or retardation coefficients.
Assuntos
Complexos Multienzimáticos/análise , Peptídeo Sintases/análise , Trifosfato de Adenosina/metabolismo , Difosfatos/metabolismo , Eletroforese em Gel de Poliacrilamida , Substâncias Macromoleculares , Peso Molecular , Ornitina/metabolismo , Dodecilsulfato de Sódio , Valina/metabolismoRESUMO
1. Chromatography on hydroxyapatite and on aminohexyl-Sepharose as well as isoelectric focusing were introduced as new effective purification procedures for phenylalanine racemase (EC 5.1.1.11). The enzyme preparations obtained were essentially homogeneous, as demonstrated by specific activity measurements and polyacrylamide gel electrophoresis. 2. The enzyme is not dissociable by sodium dodecyl sulfate. 3. Phenylalanine racemase is an acidic protein with an isoelectric point of approx. 4.6 (isoelectric focusing). 4. The Michaelis constants of L-Phe and D-Phe in the aminoacyl adenylate activation are 0.06 and 0.13 mM, respectively. 5. From our studies with structural analogues of phenylalanine we infer that the amino group of this amino acid is essential for its binding to the aminoacyl adenylate reaction center. The carboxyl group is not at all or only weakly bound. The benzene ring of phenylalanine which determines substrate recognition also seems to be of minor importance for substrate binding.
Assuntos
Isomerases de Aminoácido/metabolismo , Trifosfato de Adenosina/metabolismo , Isomerases de Aminoácido/isolamento & purificação , Bacillus/enzimologia , Difosfatos/metabolismo , Cinética , Peso Molecular , Complexos Multienzimáticos , Peptídeo Sintases , Fenetilaminas/farmacologia , Fenilalanina/metabolismo , Dodecilsulfato de Sódio , TermodinâmicaRESUMO
Alamethicin synthetase was extracted from the fungus Trichoderma viride at the end of its exponential growth phase. It is multienzyme complex with a molecular weight of approx. 480 000. The biosynthesis of alamethicin is initiated on the synthetase by acetylation of thiolester-bound aminoisobutyric acid, which remains enzyme bound. Acetyl-CoA serves as the acetate donor. Of the alamethicin constituents, glycine, alanine and valine are also acetylated when incubated alone. This acetylation is prevented by added aminoisobutyric acid, which indicates that the site on alamethicin synthetase catalyzing the acetylation has a preference for aminoisobutyric acid. Alamethicin formation on the synthetase is terminated by linkage of phenylalaninol to the carboxyl terminus of the peptide. It is unlikely that the amino alcohol is a degradation product of alamethicin or that it had been split off from the synthetase complex. Thus it is probably the reaction product of a separate enzyme system.
Assuntos
Alameticina/biossíntese , Antibacterianos/biossíntese , Fungos Mitospóricos/metabolismo , Peptídeo Sintases/metabolismo , Trichoderma/metabolismo , Acetilação , Alameticina/síntese química , Ácidos Aminoisobutíricos/metabolismo , Sítios de Ligação , Fenômenos Químicos , Química , Fenilalanina/análogos & derivados , Fenilalanina/metabolismoRESUMO
1. The cyclic peptide antibiotic tyrocidine which inhibits RNA synthesis in vitro by forming a complex with the DNA (Schazschneider, B., Ristow, H. and Kleinkauf, H. (1974) Nature 249, 757-759) induces hypochromicity of the DNA. The complex dissociates at elevated temperatures but which are below the melting temperature of the DNA. 2. The linear peptide antibiotic gramicidin which reverses the inhibitory effect of tyrocidine (Ristow, H., Schazschneider, B., Bauer, K. and Kleinkauf, H. (1975) Biochim. Biophys. Acta 390, 246-252) does not bind to DNA and does not induce hypochromicity of the DNA. However, the DNA-tyrocidine complex dissociates at lower temperatures when gramicidin is present. Thus gramicidin weakens the binding of tyrocidine to DNA. 3. The presence of DNA quenches the fluorescence of tyrocidine but not that of gramicidin. This quenching of tyrocidine fluorescence is reduced in the presence of gramicidin. 4. Tyrocidine inhibits transcription of single-stranded DNA as well. This inhibition too can be reversed by gramicidin. Thus the action of the peptides is not dependent on a double-stranded DNA conformation.
Assuntos
DNA Bacteriano , Gramicidina , Tirocidina , Tirotricina , Bacillus/metabolismo , Sítios de Ligação , Clostridium perfringens/metabolismo , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Gramicidina/farmacologia , Micrococcus/metabolismo , Desnaturação de Ácido Nucleico , Especificidade da Espécie , Espectrometria de Fluorescência , Temperatura , Transcrição Gênica/efeitos dos fármacos , Tirocidina/farmacologiaRESUMO
The boundaries of the structural domains in peptide synthetases and the conformational changes related to catalysis were investigated by limited proteolysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteolysis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain boundaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain. Limited proteolysis of an active N-terminal truncated deletion mutant, His6DeltaTY1, generated two stable and structurally independent subunits, corresponding to the subdomains of the adenylation domain. The structural integrity of the carrier domain was substantiated by its resistance to proteolytic degradation. Evidence is provided that the C-terminal "spacer" region with epimerising and/or condensing activity folds into an autonomous domain stable against degradation by limited proteoly sis. In the presence of substrates, reduced susceptibility to proteolysis was observed in the linker region connecting the subdomains of the adenylation domain, and corresponding to a peptide stretch of low electron density in the X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas the linker regions connecting the structural domains are of low homology with a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions in the phosphate chain, AMP-PcP, AMP-PNP and AMP-cPP, strongly suggests that the generation of a productive complex is associated with the ability of the beta, gamma-pyrophosphate moiety of ATP to adopt the proper active-site conformation. These data substantiate the observation that peptide synthetases undergo a series of conformational changes in the process of adenylate formation and product release.
Assuntos
Peptídeo Sintases/química , Conformação Proteica , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/fisiologia , Adenilil Imidodifosfato/química , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Catálise , Difosfatos/química , Cinética , Ligantes , Fragmentos de Peptídeos/química , Peptídeo Sintases/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Deleção de SequênciaRESUMO
Biosynthetic pathways leading to antibiotics have often been found to be clustered, and new organizational forms of multifunctional enzymes have been discovered. Such polyenzymes accomplish the synthesis of complex metabolites such as peptides or polyketides by a sequence of enzymatic reactions. So, reactions leading to the tripeptide precursor of beta-lactam antibiotics, ACV, or to the cycloundecapeptide cyclosporine have been fused into single polypeptide chain synthetases, respectively. In certain isofunctional sites restricted similarities have been detected.
Assuntos
Antibacterianos/biossíntese , Complexos Multienzimáticos/metabolismo , Clonagem Molecular , Genes , Lactamas , Família Multigênica , Peptídeo Sintases/metabolismoRESUMO
The earlier determined molecular mass of 0.8 MDa for the multifunctional polypeptide, cyclosporin synthetase, was re-evaluated by SDS-PAGE and CsCl density gradient centrifugation. In SDS-PAGE, new molecular mass values as standards were available from sequencing data. In the CsCl density gradient extremely low protein concentrations, such as 10-50 nM could be analysed due to the fluorescence detection system of the analytical ultracentrifuge. Both methods yielded approximately the same value of about 1.4 MDa. Using this molecular mass of cyclosporin synthetase as a reference the molecular masses of various related enzymes could be re-evaluated in SDS-PAGE. The sedimentation coefficient of 26.3 S for cyclosporin synthetase indicates an oblate overall shape of the enzyme.
Assuntos
Cloretos , Complexos Multienzimáticos/química , Peptídeo Sintases/química , Césio , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Peso Molecular , UltracentrifugaçãoRESUMO
The reversible denaturation of the multifunctional polypeptide, cyclosporin synthetase, by urea was analyzed. It is possible to discriminate between at least two stages of enzyme denaturation. While at low urea concentration (up to 0.8M) cyclosporin A formation is inhibited, synthesis of the diketopiperazine cyclo-(D-alanyl-N-methylleucyl), a molecule representing a partial sequence of cyclosporin A is still detectable. At higher concentrations of urea the enzyme preparation is totally inactive. This inactivation is a consequence of conformational change(s) of cyclosporin synthetase as shown by fluorescence emission spectra of native and denatured enzyme. These data imply a consecutive folding/defolding mechanism for the different domains forming the multifuntional polypeptide.
Assuntos
Complexos Multienzimáticos/química , Peptídeo Sintases/química , Ureia/farmacologia , Ciclosporina/biossíntese , Fungos/enzimologia , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/metabolismo , Peptídeos Cíclicos/biossíntese , Piperazinas/metabolismo , Conformação Proteica , Desnaturação Proteica , Dobramento de ProteínaRESUMO
Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1,629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32P]PP(i) exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.
Assuntos
Monofosfato de Adenosina/metabolismo , Coenzima A Ligases/química , Peptídeo Sintases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Coenzima A Ligases/metabolismo , Escherichia coli , Dados de Sequência Molecular , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fenilalanina/metabolismo , Fosfatos/metabolismo , Proteínas Recombinantes , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1]CyA, dihydro-CyA, [L-norvaline2,5, N-methyl-L-norvaline11]CyA, [L-allo-isoleucine5, N-methyl-L-allo-isoleucine11]CyA, [D-2-aminobutyric acid8]CyA, [beta-chloro-D-alanine8]CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide [L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8]CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.
Assuntos
Ciclosporina/biossíntese , Ciclosporinas/biossíntese , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Centrifugação com Gradiente de Concentração , Ciclosporinas/farmacologia , Eletroforese em Gel de Poliacrilamida , Imunossupressores/metabolismo , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Peptídeo Sintases/químicaRESUMO
Enzymatically formed peptides show positional variations as well as highly conserved amino acids. In the cases of gramicidin S, tyrocidine, linear gramicidins, enniatins, echinocandins and viridogrisein in vivo and in vitro studies indicate substrate selection at the level of amino acid activation as a major control step. Evidence for proof-reading steps beyond activation has been obtained in penicillin and cyclosporin biosynthesis. Activated substrate analogues may promote the formation of side products such as dipeptides and cyclodipeptides. Modifications of intermediates, such as N-methylation, influence the rates of peptide synthesis. These control steps pose limitations for the application of such enzyme systems in the production of peptide libraries. They may originate from a target oriented evolution of these synthetases.
Assuntos
Antibacterianos/biossíntese , Biossíntese Peptídica , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Ciclosporinas/biossíntese , Ciclosporinas/química , Evolução Molecular Direcionada , Gramicidina/análogos & derivados , Gramicidina/biossíntese , Gramicidina/química , Elongação Traducional da Cadeia Peptídica , Biblioteca de Peptídeos , Especificidade por SubstratoRESUMO
Beauvericin synthetase, a multifunctional enzyme catalyzing depsipeptide formation in Beauveria bassiana was purified to near homogeneity. The enzyme consists of a single polypeptide chain with a molecular mass of about 250 kdaltons. The mechanism of beauvericin formation is very similar to that of the cyclohexadepsipeptide enniatin. The constituents of the beauvericin molecule, L-phenylalanine and D-alpha-hydroxyisovaleric acid are activated as thioesters via the corresponding adenylates. N-Methylation takes place at the thioester bound stage of the phenylalanine residues. Omission of the methyl donor S-adenosyl-L-methionine results in the formation of demethylbeauvericin. Studies on substrate specificity revealed that phenylalanine could be replaced by a number of other aromatic or aliphatic amino acids like beta-phenylserine, ortho-, meta-, para-fluorophenylalanine, isoleucine, norleucine and leucine. Valine, the constituent amino acid of enniatin B was not accepted by the enzyme.
Assuntos
Antibacterianos/biossíntese , Depsipeptídeos , Peptídeo Sintases/análise , Peptídeos , Concentração de Íons de Hidrogênio , Cinética , Metilação , Fungos Mitospóricos/enzimologia , Peso Molecular , Peptídeos Cíclicos/biossíntese , Especificidade por Substrato , TemperaturaRESUMO
Enniatin synthetases from the cyclodepsipeptide producers Fusarium lateritium and Fusarium sambucinum were purified to homogeneity and characterized. Like the previously described enniatin synthetase from Fusarium scirpi both enzymes consist of a single polypeptide chain and are very similar concerning their Mr (250 kdaltons) and reaction mechanism. Limited proteolytic digests show only slight differences in their patterns in SDS-gels. Interestingly the synthetases differ in their amino acids specificities. The enzyme from the enniatin A producer F. sambucinum exhibits a high affinity to the substrate amino acids L-Leu and L-Ile. In contrast the synthetase from the enniatin B producer F. lateritium preferably accepts L-Val, the constituent amino acid of enniatin B.
Assuntos
Fusarium/enzimologia , Peptídeo Sintases/isolamento & purificação , Anticorpos Monoclonais , Peptídeo Sintases/análise , Peptídeo Sintases/imunologia , Especificidade por SubstratoRESUMO
A covalently closed circular (ccc) DNA, with a weight of 44.7 x 10(6) daltons, has been isolated from Bacillus brevis ATCC 999 (a gramicidin S producer) and from the gramicidin S-negative mutant EB16. The ccc DNA in the case of the parent strain, is mainly (99%) attached to the chromosome and membrane fraction. A restriction enzyme map of the plasmid DNA was constructed for the enzymes SalI, SmaI and BamHI, which cleaved the plasmid DNA into two, two and six fragments respectively. Further digestion with the endonucleases EcoRI and HindIII cleaved the plasmid into 17 and 22 fragments.
Assuntos
Bacillus/genética , Plasmídeos , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Membrana Celular/análise , Mapeamento Cromossômico , Cromossomos Bacterianos/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Resistência Microbiana a Medicamentos , Endonucleases/farmacologiaRESUMO
An enzyme preparation, isolated from extracts of the fungus Beauveria nivea (previously designated Tolypocladium inflatum), is able to synthesize cyclosporins (Cy's) in vitro. At suboptimal temperature it was possible to yield about 50 micrograms of CyA per ml. The enzyme also produces several of the naturally occurring congeners of CyA, such as the Cy's B, C, D, G, M, O, Q, U and V and some of the analogues known to be produced by the fungus via precursor directed biosynthesis, like dihydro-CyA, [N-methyl-L-beta-cyclohexylalanine]CyA, [L-allylglycine]CyA and [D-serine]CyA. Furthermore, Cy's not obtainable by the fungus could be prepared by the enzyme system in the presence of the appropriate precursor amino acids; the synthesis of [N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-norvaline, N-methyl-L-norvaline]CyA, [L-allo-isoleucine, N-methyl-L-allo-isoleucine]CyA, [L-allo-isoleucine]CyA, [D-2-aminobutyric acid]CyA and [beta-chloro-D-alanine]CyA could be established. The immunosuppressive effects of the new derivatives are discussed.
Assuntos
Ciclosporinas/biossíntese , Fungos Mitospóricos/enzimologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina/métodos , Técnicas In Vitro , Espectrometria de Massas , Relação Estrutura-AtividadeRESUMO
The enzymatic formation of the cyclodepsipeptide beauvericin was demonstrated in cell-free extracts from Beauveria bassiana. In analogy to the enniatin synthetase system formation of beauvericin is strictly dependent on the presence of the constituent amino and hydroxy acid, S-adenosylmethionine, and ATP/Mg2+. Synthesizing activity could be enriched about 12-fold by fractional ammonium sulfate precipitation. Besides the enniatin synthetase system this represents another example of the cell-free synthesis of a depsipeptide from eucaryotic origin.
Assuntos
Antibacterianos/biossíntese , Depsipeptídeos , Fungos Mitospóricos/metabolismo , Peptídeos , Radioisótopos de Carbono , Sistema Livre de Células , Cinética , Peptídeos Cíclicos/biossíntese , Fenilalanina/metabolismo , Valina/metabolismoRESUMO
New cyclohexadepsipeptides of the enniatin type with potential anthelmintic properties were produced by two different strategies: 1. In vitro synthesis by use of the multienzyme enniatin synthetase, and 2. in vivo precursor feeding of enniatin producing strains Fusarium scirpi and Fusarium sambucinum. The compounds were analyzed by HPLC, various NMR measurements and mass spectrometry. The three N-methyl L-amino acid positions in the enniatin B molecule could be gradually replaced by other (N-methyl) L-amino acids, e.g. alanine, cysteine, threonine and serine. The latter two amino acids yield new enniatins with functional groups in the hydrophobic side chains. Similarly the three D-2-hydroxyisovalerate residues, present in all naturally occuring enniatins, could be substituted by D-2-hydroxybutyric acid and D-lactic acid. Despite its lower yield the in vitro synthesis has the advantage of a broader variety of products formed.
Assuntos
Anti-Helmínticos/metabolismo , Antibacterianos/biossíntese , Depsipeptídeos , Fusarium/metabolismo , Peptídeos , Anti-Helmínticos/química , Antibacterianos/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Meios de Cultura , Proteínas Fúngicas/biossíntese , Fusarium/enzimologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Peptídeo Sintases/metabolismoRESUMO
The peptide part of CoA, 4'-phosphopantetheine, has been identified as an essential cofactor in in the biosynthesis of fatty acids, polyketides, depsipeptides, peptides, and compounds derived from both carboxylic and amino acid precursors, like rapamycin. The cofactor is attached to a unique protein moiety, referred to as acyl carrier protein, aminoacyl carrier protein, or peptidyl carrier protein. These carrier proteins are either associated to enzyme complexes (type II) or integrated within multifunctional polypeptide chains (type I). The cofactor is added in a post-translational modification reaction by highly specific transferases, acting on CoA. The functions of carrier proteins in directed condensation reactions are: (1) the selection of substrates to be attached as thioesters, (2) the stabilization of intermediates, (3) the presentation of intermediates for modification by associated enzyme activities, (4) facilitation of the directed condensation reactions of two adjacent intermediates, and (5) assistance in the termination reaction(s) leading to product release.
Assuntos
Ácidos Graxos/biossíntese , Panteteína/análogos & derivados , Peptídeos/metabolismo , Proteínas de Transporte/metabolismo , Coenzima A/metabolismo , Panteteína/metabolismoRESUMO
ACV synthetase forms the tripeptide precursor of penicillins and cephalosporins from alpha-aminoadipate, cysteine, and valine. Catalytic sites for substrate carboxyl activation as adenylates, peptide bond formations, epimerization and release of the tripeptide-thioester are integrated in multifunctional enzymes of 405 to 425 kD. These have been characterized from several pro- and eukaryotic beta-lactam producers. Implications of these results for the thio-template mechanism of peptide formation are discussed, as well as the use of this multienzyme as a model system for enzymatic peptide synthesis.