RESUMEN
4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.
Asunto(s)
Anticuerpos/química , Proteínas Bacterianas/química , Inmunoconjugados/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Antineoplásicos/química , Catálisis , Estructura Molecular , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Phosphopantetheine transferases (PPTases) can be used to efficiently prepare site-specific antibody-drug conjugates (ADCs) by enzymatically coupling coenzyme A (CoA)-linker payloads to 11-12 amino acid peptide substrates inserted into antibodies. Here, a two-step strategy is established wherein in a first step, CoA analogs with various bioorthogonal reactivities are enzymatically installed on the antibody for chemical conjugation with a cytotoxic payload in a second step. Because of the high structural similarity of these CoA analogs to the natural PPTase substrate CoA-SH, the first step proceeds very efficiently and enables the use of peptide tags as short as 6 amino acids compared to the 11-12 amino acids required for efficient one-step coupling of the payload molecule. Furthermore, two-step conjugation provides access to diverse linker chemistries and spacers of varying lengths. The potency of the ADCs was largely independent of linker architecture. In mice, proteolytic cleavage was observed for some C-terminally linked auristatin payloads. The in vivo stability of these ADCs was significantly improved by reduction of the linker length. In addition, linker stability was found to be modulated by attachment site, and this, together with linker length, provides an opportunity for maximizing ADC stability without sacrificing potency.
Asunto(s)
Anticuerpos Monoclonales/química , Coenzima A/química , Citotoxinas/química , Inmunoconjugados/química , Aminobenzoatos/administración & dosificación , Aminobenzoatos/química , Animales , Citotoxinas/administración & dosificación , Estabilidad de Medicamentos , Ratones , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Relación Estructura-ActividadRESUMEN
Post-translational modification catalyzed by phosphopantetheinyl transferases (PPTases) has previously been used to site-specifically label proteins with structurally diverse molecules. PPTase catalysis results in covalent modification of a serine residue in acyl/peptidyl carrier proteins and their surrogate substrates which are typically fused to the N- or C-terminus. To test the utility of PPTases for preparing antibody-drug conjugates (ADCs), we inserted 11 and 12-mer PPTase substrate sequences at 110 constant region loop positions of trastuzumab. Using Sfp-PPTase, 63 sites could be efficiently labeled with an auristatin toxin, resulting in 95 homogeneous ADCs. ADCs labeled in the CH1 domain displayed in general excellent pharmacokinetic profiles and negligible drug loss. A subset of CH2 domain conjugates underwent rapid clearance in mouse pharmacokinetic studies. Rapid clearance correlated with lower thermal stability of the particular antibodies. Independent of conjugation site, almost all ADCs exhibited subnanomolar in vitro cytotoxicity against HER2-positive cell lines. One selected ADC was shown to induce tumor regression in a xenograft model at a single dose of 3 mg/kg, demonstrating that PPTase-mediated conjugation is suitable for the production of highly efficacious and homogeneous ADCs.
Asunto(s)
Aminobenzoatos/metabolismo , Antineoplásicos/metabolismo , Proteínas Bacterianas/metabolismo , Inmunoconjugados/metabolismo , Neoplasias/tratamiento farmacológico , Oligopéptidos/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Trastuzumab/metabolismo , Aminobenzoatos/química , Aminobenzoatos/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Humanos , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Oligopéptidos/química , Oligopéptidos/uso terapéutico , Péptidos/química , Péptidos/metabolismo , Especificidad por Sustrato , Trastuzumab/química , Trastuzumab/uso terapéuticoRESUMEN
Protein interaction surface mapping using MS is widely applied but comparatively resource-intensive. Here, a workflow adaptation for use of isotope-coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible.
Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Sitios de Unión , Marcaje Isotópico , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.
Asunto(s)
Colorantes Fluorescentes/química , Lisina/análogos & derivados , Péptidos/química , Proteínas/química , Coloración y Etiquetado/métodos , Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Lisina/química , Estructura MolecularRESUMEN
Sticky residue: Pyrroline-carboxy-lysine (Pcl) can be readily incorporated into proteins expressed in E. coli and mammalian cells by using the pyrrolysyl tRNA/tRNA synthetase pair. Pcl can be used as a single amino acid purification tag and can be site-specifically modified with functional probes during the elution process.
Asunto(s)
Lisina/análogos & derivados , Proteínas/química , Proteínas/aislamiento & purificación , Benzaldehídos/química , Sitios de Unión , Lisina/química , Lisina/metabolismo , Estructura MolecularRESUMEN
The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.
Asunto(s)
Aminoácidos/inmunología , Linfocitos T CD4-Positivos/inmunología , Tolerancia Inmunológica/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Sustitución de Aminoácidos , Aminoácidos/genética , Animales , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/inmunología , Factor de Crecimiento Epidérmico/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Inmunización/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Fenilalanina/análogos & derivados , Fenilalanina/genética , Fenilalanina/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Tirosina/análogos & derivados , Tirosina/genética , Tirosina/inmunologíaRESUMEN
Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.
Asunto(s)
Lisina/química , Proteínas/química , Pirroles/química , Medios de Cultivo , Escherichia coli/genética , Resonancia Magnética Nuclear BiomolecularRESUMEN
D-ornithine has previously been suggested to enhance the expression of pyrrolysine-containing proteins. We unexpectedly discovered that uptake of D-ornithine results in the insertion of a new amino acid, pyrroline-carboxy-lysine (Pcl) instead of the anticipated pyrrolysine (Pyl). Our feeding and biochemical studies point to specific roles of the poorly understood Pyl biosynthetic enzymes PylC and PylD in converting L-lysine and D-ornithine to Pcl and confirm intermediates in the biosynthesis of Pyl.
Asunto(s)
Lisina/análogos & derivados , Ornitina/farmacología , Secuencia de Aminoácidos , Escherichia coli , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Lisina/biosíntesis , Lisina/química , Methanosarcina/genética , Methanosarcina/metabolismo , Estructura Molecular , Ornitina/química , Ornitina/metabolismo , Plásmidos , Regiones Promotoras GenéticasRESUMEN
For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.
Asunto(s)
Aminoácidos/genética , Inmunoterapia/métodos , Ingeniería de Proteínas/métodos , Autotolerancia/inmunología , Aminoácidos/inmunología , Animales , Formación de Anticuerpos , Autoanticuerpos , Autoantígenos/genética , Inmunoglobulina G , Ratones , Fenilalanina/análogos & derivados , Fenilalanina/genética , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.
Asunto(s)
Sustitución de Aminoácidos , Formación de Anticuerpos/efectos de los fármacos , Mutación Missense , Autotolerancia/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/inmunología , Endotoxemia/inducido químicamente , Endotoxemia/tratamiento farmacológico , Endotoxemia/genética , Endotoxemia/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/farmacología , Inmunoquímica , Lipopolisacáridos/toxicidad , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Nitrofenoles/inmunología , Nitrofenoles/farmacología , Autotolerancia/genética , Autotolerancia/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Vacunas/genética , Vacunas/inmunologíaRESUMEN
Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought.
Asunto(s)
Iminas/química , Oligopéptidos/química , Oxidorreductasas/química , Péptido Sintasas/química , Péptidos Cíclicos/síntesis química , Ciclización , Conformación Molecular , Péptidos Cíclicos/química , Estereoisomerismo , Factores de TiempoRESUMEN
The acidic lipopeptides, including the clinically approved antibiotic daptomycin, constitute a class of structurally related branched cyclic peptidolactones and peptidolactams synthesized by nonribosomal peptide synthetases (NRPSs). In this study, the excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, which were chemically produced by solid phase peptide synthesis. Applying this chemoenzymatic strategy, we generated derivatives of A54145 and daptomycin as well as hybrid molecules of both compounds. Bioactivity determination of the derived cyclic molecules revealed new insights into the structure-activity relationship of the acidic lipopeptide family. The general importance of several amino acid positions, including two conserved aspartic acid residues, was confirmed to be substantial for antibiotic potency. As a robust macrocyclization catalyst, the peptide cyclase excised from A54145 synthetase is the first cyclase of a branched cyclic lipopeptide, which catalyzes both macrolactonization and macrolactamization. The results presented herein illustrate the advantages of combining organic synthesis with natural product biosynthetic enzymes to explore the interplay between structural features and biological activity.
Asunto(s)
Daptomicina/química , Diseño de Fármacos , Secuencia de Aminoácidos , Catálisis , Ciclización , Daptomicina/análogos & derivados , Daptomicina/síntesis química , Lactamas Macrocíclicas/química , Lipoproteínas/síntesis química , Lipoproteínas/química , Lipoproteínas/fisiología , Pruebas de Sensibilidad Microbiana , Modelos Químicos , Datos de Secuencia Molecular , Estructura Molecular , Péptido Sintasas/química , Péptidos Cíclicos/química , Relación Estructura-ActividadRESUMEN
Non-ribosomally synthesized peptides have compelling biological activities ranging from antimicrobial to immunosuppressive and from cytostatic to antitumor. The broad spectrum of applications in modern medicine is reflected in the great structural diversity of these natural products. They contain unique building blocks, such as d-amino acids, fatty acids, sugar moieties, and heterocyclic elements, as well as halogenated, methylated, and formylated residues. In the past decades, significant progress has been made toward the understanding of the biosynthesis of these secondary metabolites by nonribosomal peptide synthetases (NRPSs) and their associated tailoring enzymes. Guided by this knowledge, researchers genetically redesigned the NRPS template to synthesize new peptide products. Moreover, chemoenzymatic strategies were developed to rationally engineer nonribosomal peptides products in order to increase or alter their bioactivities. Specifically, chemical synthesis combined with peptide cyclization mediated by nonribosomal thioesterase domains enabled the synthesis of glycosylated cyclopeptides, inhibitors of integrin receptors, peptide/polyketide hybrids, lipopeptide antibiotics, and streptogramin B antibiotics. In addition to the synthetic potential of these cyclization catalysts, which is the main focus of this review, different enzymes for tailoring of peptide scaffolds as well as the manipulation of carrier proteins with reporter-labeled coenzyme A analogs are discussed.
Asunto(s)
Péptido Sintasas/química , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Procesamiento Proteico-Postraduccional , Proteínas Portadoras/química , Péptidos Cíclicos/farmacología , Conformación Proteica , Estructura Terciaria de Proteína , Tioléster Hidrolasas/químicaRESUMEN
Macrocyclization of synthetic peptides by thioesterase (TE) domains excised from nonribosomal peptide synthetases (NRPS) has been limited to peptides that contain TE-specific recognition elements. To alter substrate specificity of these enzymes by evolution efforts, macrocyclization has to be detected under high-throughput conditions. Here we describe a method to selectively detect cyclic peptides by fluorescence resonance energy transfer (FRET). Using this method, picomolar detection limits were easily realized, providing novel entry for kinetic studies of catalyzed macrocyclization. Application of this method also provides an ideal tool to track TE-mediated peptide cyclization in real time. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE and calcium-dependent antibiotic (CDA) TE. For the latter cyclase, this approach was combined with site-directed affinity labeling, opening the possibility for high-throughput enzymatic screening.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , Péptidos Cíclicos/análisis , Tioléster Hidrolasas/metabolismo , Marcadores de Afinidad , Catálisis , Ciclización , Esterasas/metabolismo , Cinética , Péptido Sintasas/metabolismo , Péptidos Cíclicos/síntesis química , Estructura Terciaria de Proteína , Tirocidina/metabolismoRESUMEN
Streptogramin B antibiotics are cyclic peptide natural products produced by Streptomyces species. In combination with the synergistic group A component, they are "last line of defense" antimicrobial agents against multiresistant cocci. The racemization sensitivity of the phenylglycine (Phg(7)) ester is a complex challenge in total chemical synthesis of streptogramin B molecules. To provide fast and easy access to novel streptogramin antibiotics, we introduce a novel chemoenzymatic strategy in which diversity is generated by standard solid phase protocols and stereoselectivity by subsequent enzymatic cyclization. For this approach, we cloned, overproduced, and biochemically characterized the recombinant thioesterase domain SnbDE TE of the pristinamycin I nonribosomal peptide synthetase from Streptomyces pristinaespiralis. SnbDE TE catalyzes regioselective ring closure of linear peptide thioester analogues of pristinamycin I as well as stereoselective cyclization out of complex in situ racemizing substrate mixtures, enabling synthesis of Streptogramin B variants via a dynamic kinetic resolution assay. A remarkable substrate tolerance was detected for the enzymatic cyclization including all the seven positions of the peptide backbone. Interestingly, SnbDE TE was observed to be the first cyclase from a macrolactone forming NRPS which is additionally able to catalyze macrolactamization of peptide thioester substrates. An N-methylated peptide bond between positions 4 and 5 is mandatory for a high substrate turnover. The presented strategy is potent to screen for analogues with improved activity and guides our understanding of structure--activity relationships in the important class of streptogramin antibiotics.
Asunto(s)
Péptido Sintasas/metabolismo , Estreptogramina B/metabolismo , Streptomyces/enzimología , Ciclización , Cinética , Péptido Sintasas/genética , Estereoisomerismo , Estreptogramina B/química , Streptomyces/genética , Streptomyces/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismoRESUMEN
Daptomycin is a branched cyclic nonribosomally assembled acidic lipopeptide, which is the first clinically approved antibiotic of this class. Here we show that the recombinant cyclization domain of the Streptomyces coelicolor calcium-dependent antibiotic (CDA) nonribosomal peptide synthetase (NRPS) is a versatile tool for the chemoenzymatic generation of daptomycin derivatives. Linear CDA undecapeptide thioesters with single exchanges at six daptomycin-specific residues were successfully cyclized by CDA cyclase. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues yielded a daptomycin derivative that lacked only the beta-methyl group of l-3-methylglutamate. Bioactivity studies with several substrate analogues revealed a significant role of nonproteinogenic constituents for antibacterial potency. In accordance with acidic lipopeptides, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the concentration of calcium ions. Single deletions of the four acidic residues in the peptide backbone suggest that only two aspartic acid residues are essential for antimicrobial potency. These two residues are strictly conserved among other nonribosomal acidic lipopeptides and the EF-motif of ribosomally assembled calmodulin. Based on these findings CDA cyclase is a versatile catalyst that can be used to generate novel daptomycin derivatives that are otherwise difficult to obtain by chemical modification of the parental tridecapeptide to improve further its therapeutic activity.
Asunto(s)
Antibacterianos/síntesis química , Daptomicina/análogos & derivados , Ionóforos/síntesis química , Péptido Sintasas/química , Secuencia de Aminoácidos , Antibacterianos/biosíntesis , Antibacterianos/farmacología , Daptomicina/biosíntesis , Daptomicina/síntesis química , Daptomicina/farmacología , Ionóforos/metabolismo , Ionóforos/farmacología , Lipoproteínas/biosíntesis , Lipoproteínas/síntesis química , Lipoproteínas/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptido Sintasas/metabolismo , Péptidos , Streptomyces , Streptomyces coelicolor/enzimologíaRESUMEN
Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr(2) and Ser(1) residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser(1) or Thr(2) by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only l-configured Ser(1) and Thr(2) for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C(2) to C(6) as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.