Viscosinamide

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General info

Original publicationNielsen, 1999
Original sourcePseudomonas sp. DR54
Other known sources (non-putative)Pseudomonas sp. A2W4.9 (Oni, 2020)
Pseudomonas sp. ICBG1870 (Fukuda, 2021)
Pseudomonas sp. ICBG1881 (Fukuda, 2021)
Stereochemistry determined byX-ray and Marfey’s analysis (P. sp. DR54) (Geudens, personal communication)
NMR spectral matching (P. sp. A2W4.9)

Chemical properties

CASn.a.
Molecular formulaC54H96N10O15
Molecular weight1125.4 g/mol
Mono-isotopic mass1124.7057 Da (isobaric to other CLiPs, see text))
SolubilityAcetonitrile, methanol, DMSO, acetone, chloroform, ethylacetate, buffer (limited) Not soluble in milliQ water.
CMC<< 1 µM
Minimal surface tension 
3D conformationX-ray (unpublished), NMR solution structure (Acetonitrile and DPC/H2O) (Geudens, 2014, Geudens, 2019)
NMR data available in literatureAcetonitrile and DPC/H2O (Geudens, 2014, Geudens, 2019, Oni, 2020)
Acetone-d6 (Fukuda, 2021)

Introduction

Viscosinamide is a cyclic lipodepsipeptide (CLiP) belonging to the viscosin group. It is a secondary metabolite produced by non-ribosomal peptide synthetases (NRPSs) in Pseudomonas species. Viscosinamide was first isolated from rhizosphere of field-grown sugar beet plants as the causal agent for the antagonistic activity of its producing bacterium against fungi. (Nielsen, 1998, Nielsen, 1999) The CLiP appears to be somewhat rare, as no other non-putative sources were described until very recently. (Oni, 2020) This could be related to the fact that during biosynthesis, viscosinamide remains bound to the cell membrane of its producing bacteria. (Nielsen, 1999) Therefore, it is only present in low quantities in the aqueous supernatant, from which CLiPs are typically extracted.

Biological activity

Viscosinamide features antagonistic activities against Gram-positive and mycobacteria, fungi and protozoa. There is no data available concerning activity against Gram-negative bacteria. Additionally, no data is available concerning anti-cancerous or haemolytic activities. (reviewed in Geudens, 2018)

Chemical structure

The chemical structure of viscosinamide was originally determined by means of mass spectrometry, (limited) NMR and CD spectroscopy. (Nielsen, 1999) Although the absolute stereochemistry was not explicitly determined, the authors deemed it identical to the one determined for viscosin based on the almost identical positive Cotton effect CD curves for both CLiPs. Later, a crystal structure (unpublished) confirmed that the proposed stereochemistry is indeed correct. The amino acid sequence of viscosinamide is thus 3-OH C10:0 – L-Leu1 – D-Gln2 – D-aThr3 – D-Val – L-Leu5 – D-Ser6 – L-Leu7 – D-Ser8 – L-Ile9 whereby the molecule is cyclised by means of an ester bond between the C-terminal carboxylic acid and the side-chain hydroxyl moiety of Thr3. The primary structure was later also confirmed by liquid state NMR spectroscopy. (Geudens, 2014) In the same study, three new homologues of viscosinamide were described, named viscosinamides B through D. These feature minor structural variations compared to viscosinamide A, as shown in the picture below. More specifically, they possess a Val9 or Leu9 instead of Ile9, or a variation in the fatty acid tail length. These types of modifications are commonly found in Pseudomonas CLiPs and likely arise from substrate flexibility of the adenylation domain of the NRPS systems.

Schematic sequences of viscosinamide A and homologues, where shapes indicate amino acid configuration (circles = L-AA, squares = D-AA) and colors indicate amino acid polarity (green = hydrophobic, red = polar)

Interestingly, the structure of viscosinamide A is neutral under physiological conditions, which is rare in CLiPs. Indeed, the only other neutral Pseudomonas CLiP is the structurally similar pseudodesmin A. Both CLiPs differ only in the stereochemistry of Leu5, featuring either an L-Leu5 (VA) or D-Leu (PsdA). Importantly, this also implies that both CLiPs cannot be discriminated based on high resolution MS or MS/MS, since only the configuration of Leu5 differs between both lipopeptides – the compounds are isobaric. The closed viscosinamide-analogue that does have a charge is viscosin. Indeed, this latter CLiP only differs at position two, where it possesses a D-Glu2 instead of D-Gln2. This modification introduces a negative charge under physiological conditions. It is believed that the charge does have a (limited) impact on the biophysical and biological behavior (Geudens, 2017).

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 viscosinamide 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 viscosinamide.

Please note that no reference spectra in DMF are available for viscosinamide B and C. However, upon request, data is available in acetonitrile-d3. The chemical shifts of viscosinamide D are identical to those of viscosinamide A. Therefore, the reference spectra of viscosinamide A can be used for matching. However, make sure to check the identity (length and saturation) of the fatty acid moiety using e.g. mass spectrometry.

References

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.

Fukuda, et al. “Insights Into the Ecological Role of Pseudomonas spp. in an Ant-plant Symbiosis.” Front Microbiol 12 (2021): https://dx.doi.org/10.3389/fmicb.2021.621274.

Geudens, et al. “Impact of a stereocentre inversion in cyclic lipodepsipeptides from the viscosin group: a comparative study of the viscosinamide and pseudodesmin conformation and self-assembly.” ChemBioChem15, 18 (2014): https://dx.doi.org/10.1002/cbic.201402389.

Geudens, et al. “Membrane interactions of natural cyclic lipodepsipeptides of the viscosin group.” Biochimica et Biophysica Acta – Biomembranes1859, 3 (2017): https://dx.doi.org/10.1016/j.bbamem.2016.12.013.

Geudens, et al. “Conformation and dynamics of the cyclic lipopeptide viscosinamide at the water-lipid interface.” Molecules24, 12 (2019): https://dx.doi.org/10.3390/molecules24122257.

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.

Nielsen, et al. “Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54.” Journal of Applied Microbiology86 (1999).

Nielsen, et al. “Secondary metabolite- en endochitinase dependent antagonism toward plant-pathogenic microfungi of Pseudomonas fluorescens isolates from sugar beet rizosphere.” Applied and Environmental Microbiology64, 10 (1998).

Oni, et al. “Biosynthesis and Antimicrobial Activity of Pseudodesmin and Viscosinamide Cyclic Lipopeptides Produced by Pseudomonads Associated with the Cocoyam Rhizosphere.” Microorganisms8, 7 (2020): https://dx.doi.org/10.3390/microorganisms8071079.

External links

Viscosinamide A – NORINE database

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