Chemical Strategies for Visualizing and Analyzing Endogenous Nonribosomal Peptide Synthetase (NRPS) Megasynthetases

Abstract

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.

Strategies for improving fengycin production: a review

“…In a similar manner to other important non-ribosomal peptide (NRP) natural products, such as vancomycin, bleomycin, cyclosporine, and surfactin, the synthesis of fengycin is accomplished via NRPS [ 29 , 30 ]. The NRPS recognizes, activates and links amino acids in a specific order to synthesize peptides via the multicarrier thiotemplate mechanism.…”

Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains

“…Some strategies that will not be discussed in detail here include the body of work pioneered by the Ishikawa and Kakeya labs in the realm of chemical proteomics and activity-based protein proling. 110,111 Building on work that designed and synthesized clickable probes 82 and inhibitors of A domains, [55][56][57] Ishikawa and coworkers have established methods that can detect and interrogate endogenous NRPSs using ELISA [112][113][114] and in vivo techniques. 115,116 This has provided valuable insight into chemical moieties required for binding and remodeling of active site architecture, while avoiding the labor-intensive and problematic enzyme purications that many other workows require.…”

Diversity, Biosynthesis and Bioactivity of Aeruginosins, a Family of Cyanobacteria-Derived Nonribosomal Linear Tetrapeptides

“…(2) the A domain specifically recognizes the substrate amino acids to generate aminoacyl-AMP by consuming ATP, which activates the amino acid substrate [50,51], and the aminoacyl-AMP meets the pantoyl-thioglyamine (Ppant) arm tethered in the T/PCP domain and links to its free thiol group, resulting in the aminoacyl-S-carrier complex (Figure 10B) [52]; (3) the subsequent transfer of the amino-S-carrier complex from the A domain to the C domain, and the binding of the upstream carriers of aminoacyl, lipoyl CoA or peptidyl groups to form peptide bonds within the active site of the C domain (Figure 10C) [53]; (4) the TE domains typically present in the NRPS termination modules catalyze hydrolytic release or cyclization of the final products [54,55].…”