RESUMO
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
A multienzyme catalyzing the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the first free intermediate in penicillin biosynthesis, was detected in an assay measuring the formation of tripeptide from L-[U-14C]valine in the presence of L-alpha-aminoadipic acid, L-cysteine, ATP, Mg2+ ions, and dithioerythritol. Enzyme was extracted from dry mycelium using a buffer with a high glycerol concentration and thiol protective agent to stabilize enzyme activity. In five steps the enzyme was purified 118-fold. It catalyzed ATP-pyrophosphate exchange in dependence of all three constituent amino acids, and the enzyme could be amino-acylated with L-[14C]valine. The molecular weight of the protein both native (in gel filtration chromatography) and denatured (polyacrylamide gel electrophoresis) was about 220 kDa. These data suggest that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase consists of a single polypeptide chain and a multienzyme thiotemplate mechanism for the reaction sequence is postulated.
Assuntos
Aspergillus nidulans/enzimologia , Oligopeptídeos/biossíntese , Penicilinas/biossíntese , Peptídeo Sintases/fisiologia , Cromatografia , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/fisiologia , Proteínas Fúngicas/ultraestrutura , Peso Molecular , Peptídeo Sintases/isolamento & purificaçãoRESUMO
The biosynthesis of peptides in nonribosomal systems is accomplished by complex multienzymes. These multienzymes assemble the required template for the construction of each natural product in the form of linearly coupled modules. This organization principle permits the integration of multistep synthetic processes on a single macromolecule.
Assuntos
Complexos Multienzimáticos/metabolismo , Biossíntese Peptídica , Peptídeo Sintases/metabolismo , Sequência de Aminoácidos , Antibacterianos/biossíntese , Bactérias/metabolismo , Dados de Sequência Molecular , Ribossomos/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.
Assuntos
Peptídeo Sintases/metabolismo , Streptomyces/enzimologia , Aminoacil-tRNA Sintetases/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Peptídeo Sintases/isolamento & purificaçãoRESUMO
The amino acid sequences of the genes coding for four multienzyme peptide synthetases, operating by the thiotemplate mechanism are compared, to show underlying principles in the biosynthetic mechanism. Alignment with other carboxylic acid activating enzymes shows the sequences. LAY(V/I)I(Y/F)TSGT(T/S)GxPKGV and GELx(L/I)GGxG(V/I) to be involved in MgATP2-binding and adenylate formation, and two other sequences, one containing the element FxLGG(H/D)S(I/L) to be involved in covalent binding of the amino acid. As a general rule, 1000 amino acid building blocks are responsible for the incorporation of one amino acid into the nascent peptide.
Assuntos
Complexos Multienzimáticos/genética , Biossíntese Peptídica , Peptídeo Sintases/genética , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Peptide synthetases consist of linearly arranged catalytic units, which by sequence alignment show equally spaced amino-acid-activating segments/modules of 600-700 amino acid residues. The consensus sequence comprises a new class of sequence motifs which are shared by some carboxyl-activating enzymes, but which do not occur in aminoacyl-tRNA synthetases. The catalytic properties of peptide synthetases with respect to the nucleotide substrate were investigated by enzyme kinetic studies. In the activation reaction ATP may be substituted by 2'-deoxy-ATP (dATP) and 7-deazaadenosine 5'-triphosphate, substrate analogues which are not recognised by many aminoacyl-tRNA synthetases, and may thus prove useful alternative substrates in the detection of peptide synthetases within complex protein mixtures. ATP derivatives substituted at C2 are substrates, while those substituted at C8 are not, indicating a preference for the anti-conformation in substrate binding. Kinetic studies revealed that coenzyme A is a non-competitive inhibitor of the activation reaction, suggesting the presence of a second nucleotide binding site which accommodates nucleotides with phosphate in the C2' or C3' position. This substrate and inhibition profile is markedly different from that of aminoacyl-tRNA synthetases and indicative of a separate homogeneous family of carboxyl-activating enzymes.
Assuntos
Trifosfato de Adenosina/metabolismo , Bactérias/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Peptídeo Sintases/química , Peptídeo Sintases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sequência de Aminoácidos , Bactérias/genética , Sítios de Ligação , Sequência Consenso , Genes Bacterianos , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Peptídeo Sintases/genética , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Characterization of the nucleotide binding domain in peptide synthetases was approached by photoaffinity labeling of tyrocidine synthetase 1 (TY1) with 2-azidoadenosine triphosphate (2-azido-ATP). Exposure of TY1 in the presence of photolabel to irradiation with ultraviolet light resulted in a time-dependent covalent modification of the enzyme with a concomitant loss of catalytic activity. Inactivation was not observed if incubation was performed in the absence of either light or the nucleotide analogue. Specificity of labeling was indicated by the ability of 2-azido-ATP to serve as a substrate in the amino acid activation reaction. The modified protein was subjected to tryptic digestion, and the fragments labeled by the nucleotide analogue were purified by reverse-phase high-performance liquid chromatography. Sequence analysis identified three tryptic peptides corresponding to residues G373-K384, W405-R416, and L483-K494, derived from the N-terminal half of the TY1 sequence. As this region shows similarity to strongly conserved regions in other peptide synthetases and acyl-CoA synthetases, it is considered to be the region catalyzing aminoacyl adenylate formation. The identified sequences appear to define components of the nucleotide binding domain found in close proximity to the adenine ring in ATP. Conservation of primary structure and homology to other carboxyl-activating enzymes of this superfamily, including peptide synthetases, insect luciferases, and acyl-CoA synthetases, is discussed.
Assuntos
Adenina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Marcadores de Afinidade , Azidas/metabolismo , Peptídeo Sintases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cinética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Peptídeo Sintases/química , Fotoquímica , Tripsina/metabolismo , Raios UltravioletaRESUMO
Nine mutants of Penicillium chrysogenum (npe1 to npe8 and npe10) impaired in penicillin biosynthesis were screened after nitrosoguanidine mutation. Mutants npe1, npe4, npe5, npe6, npe7, npe8, and npe10 failed to synthesize significant levels of penicillin, whereas strains npe2 and npe3 synthesized about 20% of the penicillin level produced by the parental strain. Mutants npe5 and npe10 did not show alpha-aminoadipylcysteinyl-valine (ACV) synthetase activity in vitro and did not form ACV in vivo. Immunoblotting analysis of the different mutants using antibodies raised against Aspergillus nidulans ACV-synthetase showed that mutants npe5 and npe10 lacked this multienzyme protein, which in the parental strain had a molecular mass of about 420 kDa, and mutants npe2 and npe3 formed reduced level of this protein. All mutants showed normal levels of isopenicillin N synthase, as shown by Western blot analysis and enzyme assays (except npe10 that lacked this enzyme and npe2 and npe3 that formed reduced levels); npe1, npe4, npe6, npe7, npe8, and npe10 lacked isopenicillin N acyltransferase. Southern hybridizations of total DNA of the parental strain and mutants npe5, npe6, npe8, and npe10 with probes internal to the pcbAB, pcbC, and penDE genes showed that mutants npe5, npe6, and npe8 had the same arrangement of the penicillin gene cluster carrying probably point mutations, but mutant npe10 lacked the three penicillin biosynthetic genes, suggesting that it had suffered a deletion of the entire penicillin cluster. Southern hybridization with a pyrG probe as control and fingerprinting analysis of total DNA of npe10 as compared to several P.chrysogenum strains and other Penicillium and Aspergillus species, confirmed that npe10 is a deletion mutant of P. chrysogenum that had lost the penicillin biosynthetic genes.
Assuntos
Genes Fúngicos , Mutação , Penicilinas/biossíntese , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Southern Blotting , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Cinética , Peso Molecular , Família Multigênica , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/isolamento & purificação , Peptídeo Sintases/metabolismo , Mutação PuntualRESUMO
The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.