Orfamide B

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Introduction

Orfamide B is a cyclic lipodepsipeptide produced by non-ribosomal peptide synthetases in various Pseudomonas species. It belongs to a group of structurally similar CLiPs called the Orfamide group. Orfamide B was first extracted as a minor compound from Pseudomonas protegens Pf-5 which produces the groups’ namesake CLiP, orfamide A. This group also contains orfamide C (Gross, 2007), orfamides D – G (Ma, 2016), orfamide H (Ma, 2020), orfamides J – M (De Roo, 2022), poaeamides A and B (Zachow, 2015, Nguyen, 2016) and the PPZPMs (Weisshoff, 2014).

Original publicationGross, 2007
Original sourcePseudomonas protegens Pf-5 (minor compound)
Other known sources (non-putative)Pseudomonas sp. CMR5c (Ma, 2016),
Pseudomonas sp. CMR12a (De Roo, 2022),
Pseudomonas protegens PH1b (minor compound) (De Roo, 2022)
Stereochemistry determined byPeptide synthesis and NMR spectral matching (De Roo, 2022)

Chemical properties

CAS939960-35-7
Molecular formulaC63H112N10O17
Molecular weight1281.6 g/mol
Mono-isotopic mass1280.8207 Da
SolubilitySoluble in methanol, ethanol, DMF, DMSO, acetonitrile
CMCn.a.
3D conformationn.a.
NMR data available in literatureDMF-d7 (Ma, 2016) (De Roo, 2022)

Chemical structure

Analysis of the orfamide B structure was initially performed by amino acid analysis and MS/MS analysis. (Gross, 2007) Later, a more in-depth analysis was performed on orfamide B produced by Pseudomonas sp. CMR5c using genomic analysis of the producing strains’ NRPS modules and NMR spectroscopy. (Ma, 2016) Stereochemical analysis was based on a genomic similarity of the CMR5c NRPS system to that of P. protegens Pf-5. The latter produces an isoform of orfamide B, called orfamide A, for which the stereochemistry was resolved experimentally. (Gross, 2007) Later however, it was found that the originally reported stereochemistry of orfamide A contains errors and should be revised to contain a D-Leu5 instead of L-Leu5 and a 3R-hydroxy fatty acid tail rather than a 3S-hydroxy one. (De Roo, 2022) Consequently, the stereochemical make-up of orfamide B, as suggested by Ma et al., should be revised. This was shown by synthesizing orfamide B and matching the NMR spectra with those of the natural compound. (De Roo, 2022) The structure of orfamide B is 3R-OH C14:0 – L-Leu1 – D-Glu2 – D-aThr3 – D-Val4 – D-Leu5 – D-Ser6 – L-Leu7 – L-Leu8 – D-Ser9 – L-Val10 and is cyclized by means of an ester bond between the C-terminal carbonyl and the hydroxyl side chain of D-alloThr3. Orfamide B differs from orfamide A at position 4, where it contains a D-Val instead of D-aIle.

Chemical structure of orfamide B
Schematic sequence of orfamide B, where shapes indicate amino acid configuration (circles = L-AA, squares = D-AA) and colors indicate amino acid polarity (green = hydrophobic, red = polar)

Biological activity

Orfamide B has antagonistic activity against several plant-pathogenic fungi such as Magnaporthe oryzae, Phytophthora porri, Pythium ultimum and Rhizoctonia solani. (Olorunleke, 2015, Ma, 2016) It has not been tested for its activity against Gram-positive or Gram-negative bacteria. (Geudens, 2018)

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 orfamide 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 orfamide B.

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.

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.

Gross, et al. “The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters.” Chemistry & Biology14, 1 (2007): https://dx.doi.org/10.1016/j.chembiol.2006.11.007.

Ma, et al. “Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species.” Frontiers in Microbiology7 (2016): https://dx.doi.org/10.3389/fmicb.2016.00382.

Ma, Z., S. Zhang, J. Liang, K. Sun, and J. Hu. 2019. ‘Isolation and characterization of a new cyclic lipopeptide orfamide H from Pseudomonas protegens CHA0′, Journal of Antibiotics.

Nguyen, et al. “Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides.” Nature Microbiology2 (2016): https://dx.doi.org/10.1038/nmicrobiol.2016.197.

Olorunleke, et al. “Interplay between orfamides, sessilins and phenazines in the control of Rhizoctonia diseases by Pseudomonas sp. CMR12a.” Environmental Microbiology Reports7, 5 (2015): https://dx.doi.org/10.1111/1758-2229.12310.

Weisshoff, et al. “PPZPMs – a novel group of cyclic lipodepsipeptides produced by the Phytophtora alni associated strain Pseudomonas sp. JX090307 – the missing link between the viscosin and amphisin group.” Natural Products Communications9, 7 (2014).

Zachow, et al. “The novel lipopeptide poaeamide of the endophyte Pseudomonas poae RE*1-1-14 is involved in pathogen suppression and root colonization.” Molecular Plant-Microbe Interactions28, 7 (2015): https://dx.doi.org/10.1094/MPMI-12-14-0406-R.

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