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&`(P`D n c 0 _" _" v D D Precursor-directed biosynthesis of fluorinated iturin A in Bacillus subtilis
Stephen Moran,a Dilip K. Rai,b Benjamin R. Clarka and Cormac D. Murphy*a
a School of Biomolecular and Biomedical Science, Ardmore House, University College Dublin, Dublin 4, Ireland. Fax: +353 (0)1 716 1183; Tel: +353 (0)1 716 1311; E-mail: cormac.d.murphy@ucd.ie
b Centre for Synthesis and Chemical Biology, University College Dublin, Dublin 4, Ireland.
Some iturin A-producing strains of Bacillus subtilis will elaborate the novel fluorinated analogue when incubated with 3-fluoro-L-tyrosine. The activity of iturin A is dependent on the D-tyrosine residue and the presence of fluorotyrosine may result in an improvement of the biological properties of this lipopeptide. The fluorinated iturin might also be used as a probe for studying its interaction with biological membranes.
Iturins, surfactins and lichensyins are bioactive lipopeptides produced by Bacillus spp via non-ribosomal peptide synthesis. Iturins have been predominantly identified in strains of Bacillus subtilis and Bacillus amyloliquifaciens ADDIN EN.CITE Phister2004151517Phister, T. G.O'Sullivan, D. J.McKay, L. L.Identification of bacilysin, chlorotetaine, and iturin A produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize doughApplied and Environmental MicrobiologyApplied and Environmental Microbiology631-6347012004Jan0099-2240ISI:000188115300083<Go to ISI>://000188115300083 Yu2002232317Yu, G. Y.Sinclair, J. B.Hartman, G. L.Bertagnolli, B. L.Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia solaniSoil Biology & BiochemistrySoil Biology & Biochemistry955-9633472002Jul0038-0717ISI:000176977600007<Go to ISI>://000176977600007 Cho2003181817Cho, S. J.Lee, S. K.Cha, B. J.Kim, Y. H.Shin, K. S.Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03Fems Microbiology LettersFems Microbiology Letters47-5122312003Jun0378-1097ISI:000183682600008<Go to ISI>://000183682600008 1-3 and are composed of a heptapeptide cylised by a (-amino acid (iturinic acid), which has a variety of chain lengths in the R-group (Figure 1). While there is some variation in the sequence of the heptapeptide, the chirality of the constituent amino acids is conserved as LDDLLDL; the organisation of the iturin A operon indicates that L-amino acids are selected for adenylation and are subsequently epimerized. ADDIN EN.CITE Tsuge2001525217Tsuge, K.Akiyama, T.Shoda, M.Cloning, sequencing, and characterization of the iturin A operonJournal of BacteriologyJournal of Bacteriology6265-6273183212001Nov0021-9193ISI:000171577200013<Go to ISI>://000171577200013 4 The antibacterial activity of iturins is restricted to Micrococcus luteus, but they have significant antifungal activity, and the producing Bacillus strains have potential as biological control agents for plant pathogens. ADDIN EN.CITE Cho2003181817Cho, S. J.Lee, S. K.Cha, B. J.Kim, Y. H.Shin, K. S.Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03Fems Microbiology LettersFems Microbiology Letters47-5122312003Jun0378-1097ISI:000183682600008<Go to ISI>://000183682600008 3 The antifungal activity of iturins is a consequence of the formation of pores in the cell membrane, causing leakage of potassium ions. ADDIN EN.CITE Besson1984636317Besson, F.Peypoux, F.Quentin, M. J.Michel, G.Action of Antifungal Peptidolipids from Bacillus-Subtilis on the Cell-Membrane of Saccharomyces-CerevisiaeJournal of AntibioticsJournal of Antibiotics172-17737219840021-8820ISI:A1984SE14000013<Go to ISI>://A1984SE14000013 5 Iturins interact with sterols and have been shown to form aggregates in the phospholipid membrane; it has been suggested that a ternary iturin/sterol/phospholipid structure might be the bioactive unit. ADDIN EN.CITE Magetdana1994373717Magetdana, R.Peypoux, F.Iturins, a Special-Class of Pore-Forming Lipopeptides - Biological and Physicochemical PropertiesToxicologyToxicology151-174871-31994Feb0300-483XISI:A1994ND55900010<Go to ISI>://A1994ND55900010 6 Furthermore, it has been proposed that this interaction is facilitated by hydrogen bonding between the hydroxyl group of the tyrosyl residue of iturin A and the hydroxyl group of the membrane cholesterol.
Precursor-directed biosynthesis has been used to generate derivatives of naturally produced bioactive compounds by adding a precursor analogue to a culture of the producing microorganism. Depending on the substrate specificity of the biosynthetic pathway the added analogue can be recognised instead of the natural precursor due to their similar chemical structure, and is subsequently incorporated into the product. Fluorinated derivatives of biosynthetic precursors are particularly useful in this regard, since they are structurally very similar to the natural substrates. Incorporation of fluorine in place of hydrogen in biochemically relevant compounds has only a minor steric effect but, owing to fluorines electronegativity, has major electronic implications, and its presence may affect enzyme-substrate binding affinities, or alter the acidity of neighbouring functional groups. ADDIN EN.CITE Harper1994999917Harper, D. B.Ohagan, D.The Fluorinated Natural-ProductsNatural Product ReportsNatural Product Reports123-1331121994Apr0265-0568ISI:A1994NG99400001<Go to ISI>://A1994NG99400001 7 Consequently fluorinated compounds are finding increased applications in the pharmaceutical and agrochemical sectors. Thus, precursor-directed biosynthesis has been used to introduce fluorine into several important antibiotics such as erythromycin, ADDIN EN.CITE Goss2005898917Goss, R. J. M.Hong, H.A novel fluorinated erythromycin antibioticChemical CommunicationsChemical Communications3983-3985312005ISI:000231131800029<Go to ISI>://000231131800029 8 actinomycin ADDIN EN.CITE Kawashima198510110117Kawashima, A.Seto, H.Kato, M.Yasuda, A.Uchida, K.Otake, N.Preparation of Fluorinated Antibiotics Followed by F-19 Nmr-Spectroscopy .2. Fluorinated ActinomycinsJournal of AntibioticsJournal of AntibioticsJ. Antibiot. (Tokyo).J Antibiot (Tokyo)1625-1628381119850021-8820ISI:A1985AUZ3300026<Go to ISI>://A1985AUZ3300026 9 and most recently calcium-dependent antibiotic. ADDIN EN.CITE Amir-Heidari2008666617Amir-Heidari, B.Thirlway, J.Micklefield, J.Auxotrophic-precursor directed biosynthesis of nonribosomal lipopeptides with modified tryptophan residuesOrganic & Biomolecular ChemistryOrganic & Biomolecular Chemistry975-9786620081477-0520ISI:000253909000004<Go to ISI>://000253909000004 10
We adopted a similar strategy by attempting to generate halogenated iturin derivatives via precursor directed biosynthesis. Taking into account the apparent significance of tyrosine in the mechanism of action of iturin A, our main objective was to generate halogenated analogues by incubating halotyrosine with iturin-producing Bacillus spp. Bacillus subtilis CS93, which is a strain originally isolated from Pozol, a fermented dough consumed by the Mayan peoples and used as a medicinal agent to treat intestinal complaints and prevent infection in wounds, was cultured in a defined growth medium, ADDIN EN.CITE Phister2004686817Phister, T. G.O'Sullivan, D. J.McKay, L. L.Identification of bacilysin, chlorotetaine, and iturin A produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize doughApplied and Environmental MicrobiologyApplied and Environmental Microbiology631-6347012004Jan0099-2240ISI:000188115300083<Go to ISI>://000188115300083 1 to which 3-fluoro-, 3-chloro- or 3-iodo-L-tyrosine was added after 24 h (0.05 or 0.5 mmol). Control cultures, which were not supplemented with halotyrosine, were grown in parallel. The growth of the cultures was monitored via optical density measurements and aliquots (1 ml) of the culture supernatants were removed and analysed by liquid chromatography-mass spectrometry (LC-MS).
Addition of 3-fluoro-L-tyrosine to the growing culture of B. subtilis CS93 resulted in an extended lag phase. Furthermore, the length of the lag period depended on the amount of 3-fluoro-L-tyrosine added (Figure 2). The inhibition of microbial growth by 2- and 3-fluorotyrosine has been observed. ADDIN EN.CITE McCord1975565617McCord, T. J.Smith, D. R.Winters, D. W.Grimes, J. F.Hulme, K. L.Robinson, L. Q.Gage, L. D.Davis, A. L.Synthesis and Microbiological Activities of Some Monohalogenated Analogs of TyrosineJournal of Medicinal ChemistryJournal of Medicinal Chemistry26-2918119750022-2623ISI:A1975V153200006<Go to ISI>://A1975V153200006 11 Analysis of the B. subtilis CS93 supernatant by 19F nuclear magnetic resonance spectroscopy revealed that no degradation of the fluorotyrosine occurred (not shown), thus the extended lag period was most likely due to the growth of a small number of fluorotyrosine-resistant bacteria in the inoculum. This suggestion is supported by the observation that when fresh medium containing fluorotyrosine was inoculated from a culture previously exposed to the compound, no inhibition of growth was observed (Figure 2). Addition of 3-chloro-L-tyrosine to the B. subtilis CS93 resulted in complete inhibition of growth; the presence of iodotyrosine did not affect growth.
Electrospray MS analysis of supernatants from cultures incubated in the absence of 3-fluoro-L-tyrosine revealed ions at m/z 1043.5 and m/z 1057.6 representing the characteristic molecular ions of iturin A containing iturinic acids with 14 and 15 carbon atoms, respectively (Figure 3) known to be produced by this strain. ADDIN EN.CITE Phister2004686817Phister, T. G.O'Sullivan, D. J.McKay, L. L.Identification of bacilysin, chlorotetaine, and iturin A produced by Bacillus sp strain CS93 isolated from pozol, a Mexican fermented maize doughApplied and Environmental MicrobiologyApplied and Environmental Microbiology631-6347012004Jan0099-2240ISI:000188115300083<Go to ISI>://000188115300083 1 The ions m/z 1065.5 and m/z 1079.6 were also evident and are sodiated forms of the two iturin A molecular ions. In the presence of 3-fluoro-L-tyrosine, new compounds appeared, which eluted just after the iturins, and are 18 Da heavier than the iturin A molecular ions (m/z 1061.6 and m/z 1075.6; Figure 3). This is entirely consistent with the presence of a fluorine atom and strongly suggests the incorporation of 3-fluoro-D-tyrosine in place of D-tyrosine. The incorporation of fluorine into iturin A is further supported by the observation of sodium adducts at m/z 1083.4 and m/z 1097.5 (the M+1 isotope peak is labelled in Fig 3). High resolution mass spectometry performed on a Micromass LC-time-of-flight instrument revealed the masses of these adducts to be 1083.5243 and 1097.5416, respectively, which fall within the acceptable ranges of the calculated masses (1083.5251 and 1097.5407, respectively). Furthermore, LC-MS/MS analysis of the supernatants, and c o m p a r i s o n o f t h e s p e c t r a w i t h n a t u r a l i t u r i n A c o n f i r m e d t h e r e p l a c e m e n t o f t y r o s i n e w i t h f l u o r o t y r o s i n e ( E S I ) . T h e f l u o r i t u r i n s w e r e p u r i f i e d f r o m c u l t u r e s u p e r n a t a n t Q% b y r e v e r s e d p h a s e s o l i d - p h a s e e x t r a c t i o n a n d H P L C , y i e l d i n g t w o p u r i f i e d f l u o r o i t u r i n s (C-14 and C-15). While the low amount of material isolated precluded a full spectroscopic analysis, 1H and 19F NMR (d6-DMSO) proved crucial in confirming the structures of the new metabolites. 1H NMR analysis of the new metabolites gave data almost identical to those for non-fluorinated iturins, but for several crucial differences in the aromatic region. The distinctive 2H tyrosine doublets ( dH 7 . 0 1 , d , J = 8 . 4 H z ; dH 6 . 6 5 , d , J = 8 . 4 H z ) A D D I N E N . C I T E <