Manuscript in the spotlight
Muangkaew, Penthip, Vic De Roo, Lu Zhou, Léa Girard, Catherine Cesa-Luna, Monica Höfte, René De Mot, Annemieke Madder, Niels Geudens, and José C. Martins. “Stereomeric Lipopeptides from a Single Non-Ribosomal Peptide Synthetase as an Additional Source of Structural and Functional Diversification in Pseudomonas Lipopeptide Biosynthesis.” International Journal of Molecular Sciences 24, no. 18 (2023): 14302. https://www.mdpi.com/1422-0067/24/18/14302.
Cyclic lipopeptides are non-ribosomal metabolites
Cyclic Lipopeptides (CLiPs) are a remarkable class of natural compounds produced by certain bacteria, particularly Pseudomonas strains, and are gaining significant attention for their diverse biological activities, including antimicrobial properties. These molecules are characterized by their cyclic structure, consisting of a peptide chain linked to a fatty acid tail. One of the aspects that makes CLiPs truly intriguing is their structural variability.
Non-Ribosomal Peptide Synthetase (NRPS) systems are the molecular factories responsible for crafting CLiPs and other complex peptides in bacteria. These intricate systems are composed of several modules, each with distinct functions. NRPS systems consist of domains like A domains (Adenylation domains), responsible for selecting and activating specific amino acids or building blocks, and C domains (Condensation domains), which catalyse peptide bond formation between these activated building blocks.
Condensation domains play a pivotal role in the assembly of the peptide chain. They act as the master builders, guiding the sequential attachment of amino acids and fatty acid moieties to create the complex CLiP structure. The condensation domains in Pseudomonas species can be subdivided in two: LCL domains and E/C domains. LCL domains link a recruited L-amino acid to the previous L-amino acid, thereby growing the peptide chain. On the other hand, E/C (dual function Epimerization/Condensation) domains bring an element of complexity and diversity to the CLiP synthesis process. These domains can perform two critical roles: firstly, they can catalyze epimerization of the amino acid that was incorporated by the preceding module of the NRPS system, which means they change the configuration of that specific amino acid from L- to its mirror image D-configuration. Secondly, E/C domains are involved in condensation, linking the newly recruited L-amino acid to the preceding (now epimerized) D-amino acid. However, it’s worth noting that not all E/C domains are fully functional. While some are fully functional in their epimerization capacity, others are non-functional. So far, the presence of epimerization activity cannot be reliably predicted through bioinformatics.
Definitive structural elucidation of entolysins
In our pursuit to validate the structural characteristics of established and potentially novel Pseudomonas CLiPs, we also published our investigation into the CLiPs synthesized by various Pseudomonas entomophilia strains associated with plants. We also compare these CLiPs to the entolysins produced by the type strain, P. entomophilia L48T. Employing our NMR fingerprint-based methodology, we reveal a consistent pattern across all cases: the production of two distinct CLiPs, one being the predominant CLiP corresponding to entolysin A, and a minor variant analogous to entolysin B. Through comprehensive NMR analysis, we establish that both entolysins, despite sharing the same molecular mass but exhibiting different chromatographic retention profiles, share an identical planar structure. Nevertheless, a critical distinction arises in their NMR fingerprints. Using chemical synthesis, we conclusively highlighted that entolysin A and B differ solely in the configuration of a single amino acid, a variation that significantly impacts their biological activities.
While it was previously believed that structural homologues in CLiPs, originating from a single NRPS, arose from the flexibility of A domains or Cstart domains, our research unveiled a new layer of complexity. We found that the partly functional E/C domain in module 14 of the NRPS system, responsible for entolysin production, played a role in producing these configurational homologues. Specifically, it introduced an extra element of ambiguity by sometimes performing epimerization (resulting in entolysin B with D-Serine 13) and other times remaining inactive (yielding entolysin A with L-Serine 13). Bioinformatic analysis reveals a possible correlation with the composition of the flanking sequence of the N-terminal secondary histidine motif characteristic for dual-function E/C-type domains.
Finally, we also assessed the antifungal properties of entolysin A and entolysin B using a PI staining assay. Surprisingly, the minor variant, entolysin B, exhibited significantly enhanced activity against both the spores and mycelium of B. cinerea R16, as well as the spores of P. oryzae VT5M1, when compared to the major variant, entolysin A. Despite the pronounced variation in their activity levels, our observations did not reveal any synergistic effects when entolysin A and B were combined. This suggests that these two variants might share similar mechanisms of action and possibly target identical components within the fungal membrane. The subtle difference in their D/L-configuration could be influencing their membrane interactions, affecting either partitioning within the membrane or their capacity to disrupt it.