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Entolysin A & B

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General info

Original publicationVallet-Gely, 2010
Original sourcePseudomonas entomophila L48T
Other known sources (non-putative)Pseudomonas entomophila COR5 (Oni, 2019)
Pseudomonas sp. COR6 (Muangkaew, 2023),
Pseudomonas sp. COR16 (Muangkaew, 2023),
Pseudomonas sp. COR17 (Muangkaew, 2023) and
Pseudomonas sp. COW47 (Muangkaew, 2023)
Stereochemistry determined byNMR fingerprint matching (Muangkaew, 2023)

Chemical properties

Molecular formulaEntolysin A: C81H141N17O23
Entolysin B: C81H141N17O23
Molecular weightEntolysin A: 1720.0 g/mol
Entolysin B: 1720.0 g/mol
Mono-isotopic massEntolysin A: 1721.1150 Da
Entolysin B: 1721.1150 Da
SolubilityMeOH, acetonitrile/water, DMF
Minimal surface tensionn.d.
3D conformationn.d.
NMR data available in literatureEntolysin A: DMF-d7 (Oni, 2019; Muangkaew, 2023)
Entolysin B: DMF-d7 (Muangkaew, 2023)


Entolysin was first named in a study into the entomopathogenic (insecticidal) properties of its producing bacterium, P. entomophila L48. However, though entolysin is required for swarming motility of its producing organism, as described for other lipopeptides, it does not participate in the virulence of P. entomophila for Drosophila melanogaster. Two homologues (entolysin A and B) were described, though their structural difference could then not be established. Since both compounds feature an identical brute formula, the difference between both compounds was unclear. In subsequent studies, the structure of entolysin A and B could be elucidated through the use of NMR fingerprint matching, whereby it was found that both compounds are diasteriomers.

Chemical structure

Structure determination of entolysin A and B, as extracted from Pseudomonas entomophila L48, was initially performed by amino acid analysis and mass spectrometry (MALDI MS/MS). The characterization of the fatty acid  was performed separately by gas chromatography and showed the presence of a 3-hydroxy decanoic acid (3-OH C10:0) for both compounds. Amino acid analysis revealed the presence of 4x Leu and 1x Ile, along with Glx, Ser, and Val. However, using mass spectrometry, the authors were unable to discriminate Leu from Ile in the sequence, as they possess the same molecular weight (isobaric). Consequently, the structures were determined to possess either leucine or isoleucine at certain positions in the amino acid chain. The stereochemistry of entolysin A was predicted based on the bioinformatic analysis of the NRPS systems responsible for its biosynthesis. (Vallet-Gely, 2010)

In a subsequent analysis, Bode et al. used  labeling experiments with deuterated [2H9]leucine followed by tandem MS analysis to determine a corrected peptide sequence of entolysin A. (Bode, 2012) Later, NMR showed an identical structure for entolysin A, as produced by Pseudomonas sp. COR5. (Oni, 2019) However, since entolysin B was not analyzed in more detail, the structural difference between the A and B homologue remained elusive.

Finally, the full structures (including stereochemistry) of both entolysin homologues were determined by chemical synthesis and NMR fingerprint matching. (Muangkaew, 2023) Here, it was made clear the entolysin A and B feature an identical amino acid sequence, but rather differ in the configuration of Ser13.

All data together establish that the structure of the main entolysin A is 3R-OH C10:0 – L-Leu – D-Glu – D-Gln – D-Val – D-Leu – D-Gln – D-Val – D-Leu – D-Gln – D-Ser – L-Val – L-Leu – L-Ser – L-Ile, while the minor entolysin B corresponds to 3R-OH C10:0 – L-Leu – D-Glu – D-Gln – D-Val – D-Leu – D-Gln – D-Val – D-Leu – D-Gln – D-Ser – L-Val – L-Leu – D-Ser – L-Ile. Here, the amino acids making up the macrocycle are underlined. Since all analyzed entolysin producers biosynthesize entolysin A and B, the presence of a stereochemical heterogeneity between major and minor seems to be a common feature of entolysin NRPS systems. So far, this observation is unique to the entolysins.

Chemical structure of entolysin A
Schematic representation of the entolysin sequence

Entolysin A & B are diasteriomers produced by a single NRPS system

Until now, it was commonly believed that the epimerization activity exhibited by a specific E/C-domain followed a binary pattern—either it had full capability to epimerize an amino acid or none at all. Any minor compounds were attributed solely to the flexibility of A- or Cstart-domains. However, this was challenged through the characterization of entolysin A and B.

Specifically, through solid-phase peptide synthesis of a small entolysin sequence library and NMR fingerprint matching, it was demonstrated that entolysin A and B are diastereomeric counterparts, differing solely in the configuration of Ser13. This remarkable finding represents the first instance of configurational homologues being produced by a single NRPS system within a Pseudomonas strain. Additionally, this unusual characteristic is observed in multiple strains of the P. entomophila species, originating from diverse biological and geographical sources.

Furthermore, while minor compounds resulting from the flexibility of A- or Cstart-domains typically account for less than 5-10% of the major compound, the ratio between entolysin B and entolysin A is significantly higher, standing at 3:2.

Bioinformatics analysis has unveiled the presence of an E/C-domain in module 14 of the entolysin-producing strains P. entomophila L48T and COR5, which led to the prediction of D-Ser13. While this prediction aligned with experimental findings for entolysin B, peptide synthesis and NMR fingerprint matching revealed that entolysin A is a diastereomeric homologue characterized by an L-Ser13. Consequently, it is evident that the production of the minor compound, entolysin B, does not result from the flexibility of an A- or Cstart-domain, as previously assumed. Instead, it appears to be sporadically influenced by the E/C-domain in module 14 of its NRPS. Further analysis of the E/C-domain sequence alignment with Pseudomonas E/C-type domains, known for their behavior regarding epimerization, highlighted that this unusual behavior coincides with the presence of an additional histidine in the secondary histidine motif characteristic of the E/C-domain. (Muangkaew, 2023)

Biological activity

Upon their extraction from P. entomophilia L48, entolysin A and B were proposed to be responsible for its producers’ pathogenicity against insects. (Vallet-Gely, 2010) However, while the study revealed a clear role for entolysin in swarming motility of its producing organism, it was not the main virulence factor against Drosophila melanogaster. In another study, purified entolysin caused hyphal leakage in the oomycete pathogen Pythium myriotylum.

An entolysin mutant, ΔetlC, showed similar biocontrol when compared with that shown by the wild type strain in a cucumber-Pythium ultimum pathosystem. (Vallet-Gely, 2010) Entolysin is capable of suppressing the cocoyam root rot pathogen Pythium myriotylum in a dose dependent manner (Oni, 2019), while crude extracts showed activity against the ascomycete pathogen P. oryzae. (Omoboye, 2019)

Finally, the antifungal activity of entolysin A and entolysin B was quantitatively assessed with a PI staining assay. (Muangkaew, 2023) The minor compound, entolysin B, was found to be more active against spores and mycelium of B. cinerea R16 and spores of P. oryzae VT5M1 compared to the major entolysin A. Despite the difference in activity, no synergy for entolysin A and B was observed, suggesting that they may have similar modes of action and target the same components in the fungal membrane, the D/L configuration possibly modulating their membrane partitioning or membrane perturbation potential.

Mode of action

The antibacterial properties of entolysin B were independent of the presence of calcium. (Reder-Christ, 2012). No further investigations into the mode of action of entolysin were performed.

NMR fingerprint data

Recently, it was established that the planar structure and stereochemistry of CLiPs can be assessed by simple comparison to a reference. (De Roo, 2022) More specifically, by matching NMR spectra of a CLiP from a newly isolated bacterial source with those of existing (reference) CLiPs, one can determine whether they are identical or not. A detailed explanation on what NMR fingerprint matching is, and how to use it, can be found here.

Below, we provide the reference NMR data of entolysin A and B in various formats. This data is recorded in DMF-d7 at room temperature, and can be used to asses similarities of newly isolated CLiPs to entolysin A and B.


Bode, et al. “Determination of the absolute configuration of peptide natural products by using stable isotope labeling and mass spectrometry.” Chemistry – A European Journal18, 8 (2012): https://dx.doi.org/10.1002/chem.201103479.

De Roo, et al. “An Nuclear Magnetic Resonance Fingerprint Matching Approach for the Identification and Structural Re-Evaluation of Pseudomonas Lipopeptides.” Microbiology Spectrum10, 4 (2022): https://dx.doi.org/doi:10.1128/spectrum.01261-22.

Muangkaew, et al. “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, 18 (2023). https://www.mdpi.com/1422-0067/24/18/14302.

Omoboye, et al. “Pseudomonas sp. COW3 produces new Bananamide-type cyclic lipopeptides with antimicrobial activity against Pythium myriotylum and Pyricularia oryzae.” Molecules 24, 22 (2019): https://dx.doi.org/10.3390/molecules24224170.

Oni, et al. “Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium).” Environmental Microbiology (2019): https://dx.doi.org/doi:10.1111/1462-2920.14520.

Reder-Christ, et al. “Model membrane studies for characterization of different antibiotic activities of lipopeptides from Pseudomonas.” Biochimica et Biophysica Acta – Biomembranes1818, 3 (2012): https://dx.doi.org/10.1016/j.bbamem.2011.08.007.

Vallet-Gely, et al. “Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production.” Applied and Environmental Microbiology76, 3 (2010): https://dx.doi.org/10.1128/AEM.02112-09.


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