RESUMEN
Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several inâ vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurolâ S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630â nm, visualized by a prominent color change from blue to orange.
Asunto(s)
Colorimetría , Péptido Sintasas , Colorimetría/métodos , Péptido Sintasas/metabolismo , Hierro , Especificidad por SustratoRESUMEN
The fungal cyclodepsipeptides (CDPs) enniatin, beauvericin, bassianolide, and PF1022 consist of alternating N-methylated l-amino and d-hydroxy acids. They are synthesized by non-ribosomal peptide synthetases (NRPS). The amino acid and hydroxy acid substrates are activated by adenylation (A) domains. Although various A domains have been characterized thus giving insights into the mechanism of substrate conversion, little is known about the utilization of hydroxy acids in NRPSs. Therefore, we used homology modelling and molecular docking of the A1 domain of enniatin synthetase (EnSyn) to gain insights into the mechanism of hydroxy acid activation. We introduced point mutations into the active site and used a photometric assay to study the substrate activation. The results suggest that the hydroxy acid is selected by interaction with backbone carbonyls rather than by a specific side chain. These insights enhance the understanding of non-amino acid substrate activation and could contribute to the engineering of depsipeptide synthetases.
Asunto(s)
Hidroxiácidos , Péptido Sintasas , Simulación del Acoplamiento Molecular , Péptido Sintasas/metabolismo , Aminoácidos/metabolismo , Dominio Catalítico , Mutagénesis Sitio-Dirigida , Especificidad por SustratoRESUMEN
Capreomycidine (Cap) is a nonproteinogenic amino acid and building block of nonribosomal peptide (NRP) natural products. We report the formation and activation of Cap in capreomycin biosynthesis. CmnC and CmnD catalyzed hydroxylation and cyclization, respectively, of l-Arg to form l-Cap. l-Cap is then adenylated by CmnG-A before being incorporated into the nonribosomal peptide. The co-crystal structures of CmnG-A with l-Cap and adenosine nucleotides provide insights into the specificity and engineering opportunities of this unique adenylation domain.
Asunto(s)
Aminoácidos , Péptido Sintasas , Péptido Sintasas/metabolismo , Capreomicina , Especificidad por Sustrato , Péptidos/químicaRESUMEN
Nonribosomal peptide synthetases (NRPS) are organized in a modular arrangement. Usually, the modular order corresponds to the assembly of the amino acids in the respective peptide, following the collinearity rule. The WS9326A biosynthetic gene cluster from Streptomyces calvus shows deviations from this rule. Most interesting is the presence of two trans adenylation domains that are located downstream of the modular NRPS arrangement. Adenylation domains are responsible for the activation of their respective amino acids. In this study, we confirmed the involvement of the trans adenylation domains in WS9326A biosynthesis by performing gene knockout experiments and by observing the selective adenylation of their predicted amino acid substrates inâ vitro. We conclude that the trans adenylation domains are essential for WS9326A biosynthesis. Moreover, both adenylation domains are observed to have MbtH-like protein dependency. Overall, we conclude that the trans adenylation domains are essential for WS9326A biosynthesis.
Asunto(s)
Streptomyces/química , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Conformación Proteica , Streptomyces/metabolismoRESUMEN
An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Péptido Sintasas/análisis , Bibliotecas de Moléculas Pequeñas/química , Escherichia coli/enzimología , Conformación Molecular , Estructura Molecular , Péptido Sintasas/metabolismo , Péptidos/química , Péptidos/metabolismoRESUMEN
Nonribosomal peptides are a prolific source of bioactive molecules biosynthesized on large, modular assembly line synthetases. Synthetic biologists seek to obtain tailored peptides with tuned or novel bioactivities by engineering modules and domains of these nonribosomal peptide synthetases. The activation step catalyzed by adenylation domains primarily selects which amino acids are incorporated into nonribosomal peptides. Here, we review experimental protocols for probing the adenylation reaction that are applicable in natural product discovery and engineering. Several alternatives to the established pyrophosphate exchange assay will be compared and potential pitfalls pointed out. Binding pocket mutagenesis of adenylation domains has been successfully conducted to adjust substrate preferences. Novel screening methods relying on yeast surface display, for instance, search a larger sequence space for improved mutants and thus allow more substantial changes in peptide structure.
Asunto(s)
Bioingeniería , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Péptido Sintasas/química , Péptidos/química , Técnicas de Visualización de Superficie Celular/métodos , Difosfatos/metabolismo , Cinética , Dominios Proteicos , Especificidad por SustratoRESUMEN
Cahuitamycins are biofilm inhibitors assembled by a convergent nonribosomal peptide synthetase pathway. Previous genetic analysis indicated that a discrete enzyme, CahJ, serves as a gatekeeper for cahuitamycin structural diversification. Here, the CahJ protein was probed structurally and functionally to guide the formation of new analogues by mutasynthetic studies. This analysis enabled the in vivo production of a new cahuitamycin congener through targeted precursor incorporation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Oligopéptidos/metabolismo , Péptido Sintasas/metabolismo , Streptomyces/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Vías Biosintéticas , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Péptido Sintasas/química , Conformación Proteica , Streptomyces/química , Especificidad por SustratoRESUMEN
Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered Aâ domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the Aâ domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created Aâ domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant Aâ domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.
Asunto(s)
Oscillatoria/enzimología , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Azidas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oscillatoria/química , Oscillatoria/metabolismo , Péptidos Cíclicos/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Nonribosomal peptide synthetases, consisted of multiple catalytic domains, are involved in the biosynthesis of an important family of bioactive natural products in a coordinated manner. Among the functional domains, adenylation domains are specifically responsible for recognizing carboxylic acid building blocks and synthesizing aminoacyl adenylates. Given their critical roles in the biosynthesis of the growing peptide, A-domains are also referred to as the "gatekeeper". In this review, very recent developments on the A-domains from NRPSs are reviewed to expand the fundamental knowledge of the A domain, including knowledge on the structures, functions, and molecular interactions. Several recent examples were also discussed to highlight the great potential of A-domain engineering. This study should provide a framework for the combinatorial biosynthesis or synthetic biology-driven microbial production of novel nonribosomal peptides.
Asunto(s)
Péptido Sintasas , Péptidos , Dominio Catalítico , Péptido Sintasas/genética , Péptido Sintasas/química , Péptido Sintasas/metabolismo , Péptidos/química , Adenosina MonofosfatoRESUMEN
Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was implemented in the NRPSpredictor and in a first step uses the physicochemical fingerprint of the 34 residues lining the active site of the phenylalanine-adenylation domain of gramicidin synthetase A [PDB ID 1 amu] to derive a feature vector. Homologous positions were extracted for A-domains with known and unknown substrate specificities and turned into feature vectors. At the same time, A-domains with known specificities towards similar substrates were clustered by physicochemical properties of amino acids (AA). In a second step, support vector machines (SVMs) were optimized from feature vectors of characterized A-domains in each of the resulting clusters. Later, SVMs were used in the variant of TSVMs that integrate a fraction of uncharacterized A-domains during training to predict unknown specificities. Finally, uncharacterized A-domains were scored by each of the constructed alignment-free models (TSVM) representing each substrate specificity resulting from the clustering. The model producing the largest score for the uncharacterized A-domain assigns the substrate specificity to it (Rausch et al., Nucleic Acids Res 33:5799-5808, 2005).