Termination of the structural confusion between plipastatin A1 and fengycin IX
Graphical abstract
Introduction
Plipastatin A1 is a macrocyclic depsilipopeptide which was first isolated by Umezawa et al. in 1986 from Bacillus cereus BMG3O2-fF67 as a phospholipase A2 inhibitor.1, 2, 3, 4 In the same issue of the journal, Jung et al. independently reported fengycin from Bacillus subtilis strain F-29-3 as an antibiotic lipopeptide.5 Although the final structure was not disclosed in that report, they obtained the identical amino acids and the fatty acid to those from plipastatin A1 by the acid hydrolysis. The structure of fengycin was revealed in 1999 by Budzikiewicz, that it is a diastereomer of plipastatin A1 (named fengycin IX) possessing d-Tyr4 and l-Tyr10 residues,6 while plipastatin A1 has the reversed permutation, l-Tyr4 and d-Tyr10.8 Due to the promising antibiotic activity of this substance and empirical safeness of the producer Bacillus subtilis, fengycin is expected to be a novel biocontrol7 to produce more than 220 scientific papers and counting. Some of those papers dealt with the biogenesis of fengycins8, 9, 10, 11, 12, 13, 14, 15, 16, 17 to disclose NRP (non-ribosormal-peptide) synthase clusters. Those involved sequences suggesting racemases. However, positions of the expected racemases accorded with plipastatin A1 in spite of fengycin producers.18, 19 The enzyme cluster which rationally explains Budzikiewicz’s fengycin IX has not so far been reported. These facts have brought structural confusion between these molecules. Although many papers followed Budzikiewicz’s structure as fengycin IX, considerable number of recent reports mentioned that these are identical compounds15, 20, 21, 22 supposedly in order to avoid the contradiction between the structure and the biogenesis. However, there was no experimental evidence. The confusion has become more serious, because at least five other structures exist as fengycin IX (some structures did not match up to their own discussions).8, 9, 10, 11, 12, 13, 14, 16, 23, 24, 25 The present studies experimentally proved that plipastatin A1 is the K+ salt whereas fengycin IX is the free form or the TFA salts. Although these molecules gave considerably different 1H NMR spectra, the quite similar 1H NMR spectrum to that of fengycin IX was changed to provide nicely accorded spectrum to that of plipastatin A1 when the sample was converted into the K+ salt. Our structural studies led a conclusion that the structures of these compounds should be settled into that of plipastatin A1 by Umezawa.
Section snippets
Results and discussions
Bacillus subtilis H336B was found to produce an antifungal cyclic peptide 1 which showed the same molecular weight 1462 as both fengycin IX and plipastatin A1 by ESIMS [1463.8019, (calcd for 1463.8038, [M+H]+ C72H112N12O20)]. Isolation was performed by a series of conventional chromatographies (XAD-7, ODS MPLC, and ODS HPLC). Analyses involving Maefey’s configurational determination26 after acidic hydrolyses disclosed l-Glx (×3), l-Orn, l-Tyr, d-Tyr, d-allo-Thr, d-Ala, l-Pro, l-Ile, and
Conclusion
We succeeded in disclosing that fengycin IX and plipastatin A1 are identical compounds although these had been considered as diastereomers at the two Tyr residues. Although reported their NMR spectra showed discordance, it could be explained by their forms; plipastatin is the K+ salt, while fengycin is the free form or the TFA salts. The present studies disclosed that structures of these molecules should be settled into that of plipastatin A1 by Umezawa. This structure showed more advantageous
Fermentation and isolation
Bacillus subtilis H336B was isolated as a contaminant from a fungal culture and was identified through BLAST search of Genbank based on 16S rRNA gene sequence. It was deposited at the Japan Collection of Microorganisms of Riken Bioresource Center as JCM 18293. The bacterium was cultured in a medium prepared from glycerol (100 g), meat extract (20 g), polypeptone (20 g), yeast extract (40 g), NaCl (8.0 g), MgSO4·7H2O (2.0 g), K2HPO4 (2.0 g), CaCO3 (12.8 g), and H2O (4.0 L) under shaking conditions (110
Acknowledgements
The present work was supported in part by the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS). The authors appreciated Emeritus Professor Haruo Seto of University of Tokyo for his kind suggestions.
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