Putisolvin III – V

General info

Original publication	Oni, 2020
Original source	Pseudomonas sp. COR19 (Oni, 2020; Muangkaew, 2024) Pseudomonas sp. NNC7(Oni, 2020; Muangkaew, 2024) Pseudomonas vlassakiae WCU_64 (Oni, 2020; Muangkaew, 2024)
Other known sources (non-putative)	Pseudomonas putida PCL1445 (Kuiper, 2003) Pseudomonas putida 267 (Kruijt, 2009) Pseudomonas putida E41 (Bernat, 2019) Pseudomonas capeferrum HN2-3 (Sheng, 2024) Pseudomonas sp. COR55 (Muangkaew, 2024) Pseudomonas vlassakiae WCU_60 (Muangkaew, 2024) Pseudomonas vlassakiae WCU_64 (Muangkaew, 2024) Pseudomonas capeferrum WCS358T (Muangkaew, 2024) Pseudomonas fulva LMG 11722T (Muangkaew, 2024)
Stereochemistry determined by	Chemical synthesis and NMR fingerprinting (Muangkaew, 2024)

Chemical properties

CAS	n.a.
Molecular formula	Putisolvin III: C65H113N13O19 Putisolvin IV: C66H115N13O19 Putisolvin V: C66H115N13O19
Molecular weight	Putisolvin III: 1380.7 g/mol Putisolvin IV: 1394.7 g/mol Putisolvin V: 1394.7 g/mol
Mono-isotopic mass	Putisolvin III: 1379.8276 Da Putisolvin IV: 1393.8432 Da Putisolvin V: 1393.8432 Da
Solubility	n.a.
CMC	n.a.
Minimal surface tension	n.a.
3D conformation	n.a.
NMR data available in literature	Muangkaew, 2024

Introduction

Putisolvin III – V were first described in the context of a study comparing Pseudomonas and cyclic lipopeptide (CLiP) diversity in the rhizosphere of a cocoyam root rot disease (CRRD) suppressive soil in Boteva, Cameroon with those from four conducive soils in Cameroon and Nigeria. (Oni, 2020)

Chemical structure

The structure of putisolvin III was initially determined by 2D NMR spectroscopy. The CLiP consists of a fatty acid linked to a peptide chain composed of 12 amino acids. The sequence was confidently established through amino acid residue type annotation using 2D TOCSY, followed by confirmation via 2D ROESY and 1H-{13C} gHMBC spectra. Notably, the participation of Ser9 and Ser12 in ester bond formation was confirmed by a distinct ³J_CH cross-peak connecting Ser9 Hβ1,2 and Ser12C’ in the ¹H-{¹³C} gHMBC spectrum. This observation aligns with the substantial downfield shift of both Ser9Cβ and Hβ1,2 resonances. Mass spectrometric data further confirmed the presence of an N-capping hexanoic fatty acid moiety (C6:0).

Minor compounds were identified as new members of the putisolvin group. These CLiPs, named putisolvin IV and V, differ from putisolvin III at position 11, where they possess an Ile11 or Leu11 respectively, compared with Val11 in the main compound. Such Ile/Val congeners are a common outcome of A-domain substrate flexibility.

Summarizing, putisolvins are CLiPs which possess a peptide moiety of 12 amino acids, whereby cyclization occurs between the side chain of Ser9 and the C-terminal carbonyl. The structure of putisolvin III is shown to be C6 : 0 – L-Leu –D-Glu – D-Leu – L-Leu – D-Gln – D-Ser – D-Val – D-Leu – D-Ser – L-Leu – L-Val – L-Ser. (Muangkaew, 2024) Remarkably, the fatty acid chain lacks the 3-hydroxyl functionality, typifying most Pseudomonas CLiPs.

Muangkaew et al. showed that the structures of the originally described putisolvin I and II contained errors, and that they should be revised. (Muangkaew, 2024) More so, the structures of putisolvin I and II were found to be identical to those of putisolvin III and IV, respectively.

Putisolvin II is isobaric to cocoyamide/gacamide, as both compounds have C₆₆H₁₁₅N₁₃O₁₉ as chemical formula. Hence, they cannot be discriminated based on mass spectrometry alone. Despite the identical chemical formula and molecular mass, both types of lipopeptides are structurally highly distinct, as they differ in amino acid sequence (both identity and length), size of the macrocycle and their fatty acid moiety.

Biological activity

For putisolvins produced by P. putida PCL1445, no antibacterial effects were observed against Pseudomonas fluorescens and Pseudomonas aeruginosa. (Kuiper et al., 2004)

At concentrations of 20–25 mg mL^-1, putisolvin immobilizes zoospores from different oomycetes and cause rapid lysis of entire zoospore populations . (Kruijt, 2009; Tran, 2008) Similarly, in vitro assays showed that putisolvin I was able to lyse a zoospore suspension of Phytophthora capsici at concentrations above 10 µM. Furthermore, an in planta assay demonstrated that a 50 μM treatment of putisolvin on a zoospore suspension of Phytophthora capsici led to a significant reduction in Phytophthora blight disease in cucumber plants. (Sheng, 2024)

Putisolvins produced by P. putida inhibited biofilm formation by P. aeruginosa PA14 and P. fluorescens WCS365 (Kuiper et al., 2004).

NMR fingerprint data

It was demonstrated that the planar structure and stereochemistry of CLiPs can be assessed by simple comparison to a reference. (De Roo, 2022; Muangkaew, 2024) 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 putisolvin I and II. This data is recorded in DMF-d7 at 333K (60°C), and can be used to asses similarities of newly isolated CLiPs to putisolvin III, IV and IV.

References

Bernat, et al. “Characterization of extracellular biosurfactants expressed by a Pseudomonas putida strain isolated from the interior of healthy roots from Sida hermaphrodita grown in a heavy metal contaminated soil.” Current Microbiology76, 11 (2019): https://dx.doi.org/10.1007/s00284-019-01757-x.

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.

Kruijt, et al. “Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.” Journal of Applied Microbiology107, 2 (2009): https://dx.doi.org/10.1111/j.1365-2672.2009.04244.x.

Kuiper, et al. “Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms.” Molecular Microbiology51, 1 (2003): https://dx.doi.org/10.1046/j.1365-2958.2003.03751.x.

Muangkaew, et al. “Breaking Cycles: Saponification-Enhanced NMR Fingerprint Matching for the Identification and Stereochemical Evaluation of Cyclic Lipodepsipeptides from Natural Sources.” Chemistry – A European Journal (2024): https://dx.doi.org/https://doi.org/10.1002/chem.202400667.

Oni, et al. “Cyclic lipopeptide-producing Pseudomonas koreensis group strains dominate the cocoyam rhizosphere of a Pythium root rot suppressive soil contrasting with P. putida prominence in conducive soils.” Environmental Microbiology 22, 12 (2020): https://dx.doi.org/10.1111/1462-2920.15127.

Sheng, et al. “The biocontrol roles of cyclic lipopeptide putisolvin produced from Pseudomonas capeferrum HN2-3 on the Phytophthora blight disease in cucumbers.” Journal of Plant Diseases and Protection 131, 2 (2024): https://dx.doi.org/10.1007/s41348-024-00874-5.