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The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nonribosomal peptide synthetases (NRPSs) biosynthesize nonribosomal peptide (NRP) natural products, which belong to the most promising resources for drug discovery and development because of their wide range of therapeutic applications. The results of genetic, biochemical, and bioinformatics analyses have enhanced our understanding of the mechanisms of the NRPS machinery. A major goal in NRP biosynthesis is to reprogram the NRPS machinery to enable the biosynthetic production of designed peptides. Reprogramming strategies for the NRPS machinery have progressed considerably in recent years, thereby increasing the yields and generating modified peptides. Here, the recent progress in NRPS reprogramming and its application in peptide synthesis are described., (© 2023 European Peptide Society and John Wiley & Sons Ltd.)

Many amino acid-containing natural products are biosynthesized by large, multifunctional enzymes known as non-ribosomal peptide synthetases (NRPSs). Adenylation (A) domains in NRPSs are responsible for the incorporation of amino acid building blocks and can be considered as engineering domains; therefore, advanced techniques are required to not only rapidly verify expression and folding, but also accelerate the functional prediction of the A-domains in lysates from native and heterologous systems. We recently developed activity-based protein profiling (ABPP) of NRPSs that offers a simple and robust analytical platform for A-domains and provides insights into their enzyme-substrate specificity. In this chapter, we describe the design and synthesis of these ABPP probes and provide a summary of our work on the development of a series of protocols for labeling, visualizing, and analyzing endogenous NRPSs in complex biological systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We present an in vitro and in-cell activity-based protein profiling (ABPP) protocol for endogenous nonribosomal peptide synthetases (NRPSs). This protocol enables the fluorescence labeling and imaging of an endogenous SrfAB-NRPS with high selectivity and sensitivity in the surfactin producer Bacillus subtilis . While we optimized this protocol for use with B. subtilis , the protocol can be applied to Aneurinibacillus migulanus and Escherichia coli . For complete details on the use and execution of this protocol, please refer to Ishikawa et al. (2022)., Competing Interests: The authors declare no competing financial interests., (© 2022 The Authors.)

Much of our current knowledge on nonribosomal peptide synthetases (NRPSs) is based on studies in which the full NRPS system or each protein domain is expressed in heterologous hosts. Consequently, methods to detect the endogenous activity of NRPSs, under natural cellular conditions, are needed for the study of NRPS cell biology. Here, we describe the in vivo activity-based protein profiling (ABPP) for endogenous NRPSs and its applications to the study of their activities in bacteria. Remarkably, in vitro and in vivo ABPP in the context of the surfactin producer Bacillus subtilis enabled the visualization, tracking, and imaging of an endogenous SrfAB-NRPS with remarkable selectivity and sensitivity. Furthermore, in vivo, ABPP allowed the discovery of the degradation processes of the endogenous SrfAB-NRPS in the context of its native producer bacteria. Overall, this study deepens our understanding of the properties of NRPSs that cannot be addressed by conventional methods., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The gatekeeping adenylation (A) domain of the non-ribosomal peptide synthetase (NRPS) selectively incorporates specific proteinogenic/non-proteinogenic amino acid into a growing peptide chain. The EntE of the enterobactin NRPS is a discrete aryl acid A-domain with 2,3-dihydroxybenzoic acid (DHB) substrate specificity. Reprogrammed EntE N235G variant possesses an enlarged substrate recognition site, and is capable of accepting non-native aryl acids. Biochemical characterization of this unique substrate recognition site should provide a better understanding of activi-site microenvironments. Here, we synthesized a non-hydrolysable adenylate analogue with 2-aminobenzoic acid (2-ABA), 3-aminobenzoic acid (3-ABA), and 4-aminobenzoic acid (4-ABA) and used them to calculate the apparent inhibition constants (K i app. ). Dose-response experiments using 3-ABA-sulfamoyladenosine (AMS) provided K i app. values of 596 nM for wild-type EntE and 2.4 nM for the N235G variants. These results suggest that 3-amino group of benzoic acid plays an important role in substrate recognition by the N235G variant. These findings would help designing aryl acid substrates with substituents at the 2- and 3-positions.

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., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aryl acids are most commonly found in iron-scavenging siderophores but are not limited to them. The nonribosomal peptide synthetase (NRPS) codes of aryl acids remain poorly elucidated relative to those of amino acids. Here, we defined more precisely the role of active-site residues in aryl acid adenylation domains (A-domains) by gradually grafting the NRPS codes used for salicylic acid (Sal) into an archetypal aryl acid A-domain, EntE [specific for the substrate 2,3-dihydroxybenzoic acid (DHB)]. Enzyme kinetics and modeling studies of these EntE variants demonstrated that the NRPS code residues at positions 236, 240, and 339 collectively regulate the substrate specificity toward DHB and Sal. Furthermore, the EntE variants exhibited the ability to activate the non-native aryl acids 3-hydroxybenzoic acid, 3-aminobenzoic acid, 3-fluorobenzoic acid, and 3-chlorobenzoic acid. These studies enhance our knowledge of the NRPS codes of aryl acids and could be exploited to reprogram aryl acid A-domains for non-native aryl acids.

Nonribosomal peptide (NRP) natural products are among the most promising resources for drug discovery and development, owing to their wide range of biological activities and therapeutic applications. These peptide metabolites are biosynthesized by large multienzyme machinery known as NRP synthetases (NRPSs). The structural complexity of a number of NRPs poses an enormous challenge in their synthesis. A major issue in this field is reprogramming NRPS machineries to allow the biosynthetic production of artificial peptides. NRPS adenylation (A) domains are responsible for the incorporation of a wide variety of amino acids and can be considered as reprogramming sites; therefore, advanced methods to accelerate the functional prediction and assessment of A-domains are required. This Concept article demonstrates that activity-based protein profiling of NRPSs offers a simple, rapid, and robust analytical platform for A-domains and provides insights into enzyme-substrate candidates and active-site microenvironments. It also describes the background associated with the development and application of a method to analyze endogenous NRPS machinery in its natural environment., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid A domains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Derivatization of a 5'-(vinylsulfonylaminodeoxy)adenosine scaffold with a clickable functionality provided an activity-based probe that was used to label native carrier protein (CP) motifs in nonribosomal peptide synthetases (NRPSs). When coupled with a fluorescent tag, this probe selectively targeted phosphopantetheinylated CPs (holo-form) from recombinant NRPS enzyme systems and in whole proteomes.

We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs.

Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5'-O-N-(aminoacyl)sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production.

We describe a general strategy for selective chemical labeling of individual adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using active site-directed proteomic probes coupled to the 5'-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.

We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5'-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated L-Phe-AMS (L-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates L-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)