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Massetolide
General info
| Original publication | Gerard, 1997 |
| Original source | Pseudomonas sp. MK90E85 (Massetolides A – D) Pseudomonas sp. MK91CC8 (Massetolides E – H) Pseudomonas sp. MK91CC8 (Massetolides I – K; feeding experiments) Pseudomonas sp. SS101 (Massetolide L) |
| Other known sources (non-putative) | Pseudomonas sp. SS101 (De Souza, 2003) |
| Stereochemistry determined by | Chiral GC (Gerard, 1997) |
Chemical properties of massetolide A
| Molecular formula | C55H97N9O16 |
| Molecular weight | 1140.4280 g/mol |
| Mono-isotopic mass | 1139.7053 Da (isobaric to other CLiPs, see text) |
| Solubility | Methanol, acetone, acetonitrile, DMF |
| CMC | 22 µM (De Souza, 2003) |
| Minimal surface tension | n.a. |
| 3D conformation | n.a. |
| NMR data available in literature | Acetone-d6 (Gerard, 1997) Methanol-d4 (De Bruijn, 2008) |
Introduction
Massetolides A – H initially were isolated from cultures of Pseudomonas sp. isolated from the surface of an unidentified leafy red algea or marine tube worm in the context of a screening program for new antimicrobial agents. (Gerard, 1997). In the same study, massetolides I – K were obtained by feeding the producing bacterium non-proteinogenic amino acids. Finally, massetolide L was characterized as a minor compound of Pseudomonas sp. SS101, which produces massetolide A as main compound. (De Bruijn, 2008)
Massetolide-producing Pseudomonas species have been found in very diverse environments: while the original bacteria were isolated from marine environments, Pseudomonas sp. SS101 was extracted from the wheat rhizosphere. Additionally, Behsaz et al. (Behsaz, 2020) reported the presence of massetolide in human stool samples, likely produced by Pseudomonas species therein.
Biological activity
Massetolide A possesses clear antagonistic activities against Gram-positive bacteria, mycobacteria, fungi and protozoa. No information is available concerning activity against Gram-negative bacteria or cancer cells. (Geudens, 2018) Interestingly, despite its hemolytic activity, a single injection of 10 mg/kg of massetolide A was reported to be non-toxic to mice. (Gerard, 1997)
Chemical structure
Massetolide A is the main compound produced by Pseudomonas sp. MK90E85, isolated from an unidentified leafy red algae . Massetolides B – D were described as minor compounds produced by this bacterium. Detailed NMR analysis on massetolide A allowed to determine the amino acid sequence, the presence of the macrocycle between Thr3 CHβ and Ile9 C’, and the presence of the N-terminal fatty acid moiety. Hydrolysis of massetolide A followed by chiral GC analysis confirmed the amino acids’ configuration. Summarizing, the structure of massetolide A is 3-OH C10:0 – L-Leu1 – D-Glu2 – D-aThr3 – D-aIle4 – L-Leu5 – D-Ser6 – L-Leu7 – D-Ser8 – L-Ile9. Structurally, this CLiPs belong to the viscosin group. It’s closed relatives are viscosin, which features a D-Val4 instead of D-allo-Ile4 and pseudophomin A, which differs only in the stereochemistry of Leu5. Since the latter only features a difference in configuration, its brute formula – and therefore, its molecular mass – are identical. They are isobaric, and cannot be discriminated based on mass spectrometric techniques alone.
Using a similar characterization methodology as used for the main compound, the minor compounds massetolide B and C only differ in the fatty acid moiety, featuring a 3-hydroxyl undecanoic (3-OH C11:0) and 3-hydroxyl dodecanoic fatty acid (3-OH C12:0) moiety respectively, where the main compound (massetolide A) possesses a 3-hydroxy decanoic acid (3-OH C10:0). massetolide D differs from the main compound at position 9, featuring a L-Leu9 rather than an L-Ile9.
Pseudomonas sp. MK91CC8, isolated from a marine tube worm, produces viscosin as it main CLiP. However, several minor compounds were also described, named massetolides E and H. They each differ only in a single residue compared to the main compound. Massetolide E and F differ only at the C-terminal residue, featuring a L-Val9 and L-Leu9 whereas the main compound viscosin possesses a L-Ile9. Massetolides G and H differ only in the fatty acid moiety, featuring a 3-hydroxyl undecanoic (3-OH C11:0) and 3-hydroxyl dodecanoic fatty acid (3-OH C12:0) moiety respectively, whereas the main compound (viscosin) possesses a 3-hydroxy decanoic acid (3-OH C10:0).
Gerard et al. performed feeding experiment using Pseudomonas sp. MK91CC8 whereby four non-proteinogenic analogues of valine and leucine (butyrine, norvaline, tert-leucine, and cyclopropylalanine) were added independently to culture media. Summarizing, the experiments with D-butyrine, D-norvaline, L- and D-tert-leucine gave no evidence for the production of new analogues. Addition of L-butyrine, L-norvaline, or L-cyclopropylalanine each gave evidence for production of new massetolide analogues that incorporated the unnatural amino acids. More specifically, three new variants were characterised: Massetolide I featured a L-butyrine residue instead of valine at position 4; In massetolide J, a norvaline residue had replaced the L-leucine residue at position 1; and finally, massetolide K represents a analogue where a norvaline residue replaced the L-leucine residue found at position 1 in viscosin.
Finally, massetolide L was characterized as a minor compound of Pseudomonas sp. SS101, which produces massetolide A as main compound. This compound features a C-terminal L-Val9 instead of L-Ile9 in massetolide A. (De Bruijn, 2008)
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 massetolides 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 the various massetolides.
Please note: The chemical shifts of massetolide B and C are identical to those of massetolide A. Please use the reference spectra of massetolide A, but check the identity (length) of the fatty acid chain by means of mass spectrometry.
Please note: the chemical shifts of massetolide E are currently not available. Consequently, not reference spectra can be given.
References
Behsaz, et al. “Peptide Sequencing Reveals Many Cyclopeptides in the Human Gut and Other Environments.” Cell Systems10, 1 (2020): https://dx.doi.org/10.1016/j.cels.2019.11.007.
De Bruijn, et al. “Massetolide A biosynthesis in Pseudomonas fluorescens.” Journal of Bacteriology190, 8 (2008): https://dx.doi.org/10.1128/JB.01563-07.
De Roo, et al. “An nuclear magnetic resonance fingerprint matching approach for the identification and structural re-evaluation of Pseudomonas lipopeptides.” Microbiology Spectrum 0, 0
https://dx.doi.org/doi:10.1128/spectrum.01261-22.
De Souza, et al. “Biochemical, genetic, and zoosporicidal properties of cyclic lipopeptide surfactants produced by Pseudomonas fluorescens.” Applied and Environmental Microbiology69, 12 (2003): https://dx.doi.org/10.1128/aem.69.12.7161-7172.2003.
Gerard, et al. “Massetolides A-H, antimycobacterial cyclic depsipeptides produced by two Pseumonads isolated from marine habitats.” Journal of Natural Products60 (1997).
Geudens, et al. “Cyclic lipodepsipeptides from Pseudomonas spp. – Biological Swiss-Army Knives.” Frontiers in Microbiology9, 1867 (2018): https://dx.doi.org/10.3389/fmicb.2018.01867.
External links
Massetolide A – NORINE database
Massetolide B – NORINE database
Massetolide C – NORINE database
Massetolide D – NORINE database
Massetolide E – NORINE database
Massetolide F – NORINE database
Massetolide G – NORINE database