Adenylation Domains in Nonribosomal Peptide Engineering

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.     

The A9 Core Sequence from NRPS Adenylation Domain Is Relevant for Thioester Formation

The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two-step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A-domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A-domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild-type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a "clip" for the proper positioning of residues important for the interaction with the T-domain, and/or stabilisation of the thioester-forming conformation.     

A Multiple‐Labeling Strategy for Nonribosomal Peptide Synthetases Using Active‐Site‐Directed Proteomic Probes for Adenylation Domains

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.     

A Competitive Enzyme‐Linked Immunosorbent Assay System for Adenylation Domains in Nonribosomal Peptide Synthetases

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.     

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains

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.     

Nonribosomal peptide synthesis in Schizosaccharomyces pombe and the architectures of ferrichrome-type siderophore synthetases in fungi

A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N-acetyl-N-hydroxy-L-ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L-N5-ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains.     

The Role of a Nonribosomal Peptide Synthetase in l‐Lysine Lactamization During Capuramycin Biosynthesis

Capuramycins are one of several known classes of natural products that contain an l ‐Lys‐derived l ‐α‐amino‐?‐caprolactam (l ‐ACL) unit. The α‐amino group of l ‐ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP‐independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP‐dependent amide‐bond‐forming catalyst responsible for the biosynthesis of the remaining amide bond present in l ‐ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l ‐Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS‐mediated mechanism is employed in the biosynthesis of other l ‐ACL‐containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS‐derived peptides that contain an unmodified l ‐Lys residue.