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Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry
Biochimica et Biophysica Acta 1438 (1999) 273^280 www.elsevier.com/locate/bba
Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry Michail M. Yakimov a
a; b;
*, Wolf-Rainer Abraham a , Holger Meyer a , Laura Giuliano c , Peter N. Golyshin a
Department of Microbiology, GBF National Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany b CSRAFA Sicilian Center for Atmospheric Research and Environmental Physics c/o Dipartamento di Fisica della Materia, Universita¨ di Messina, Salina Sperone 31, 98166 Messina, Italy c Dipartamento di Biologia Animale ed Ecologia Marina, Universita' di Messina, Salina Sperone 31, 98166 Messina, Italy Received 30 November 1998; received in revised form 18 March 1999; accepted 24 March 1999
Abstract The structural characterization of the cyclic lipoheptapeptide surfactant lichenysin A components, produced by Bacillus licheniformis strains via the non-ribosomal pathway on a corresponding peptide synthetase, was carried out using a tandem mass spectrometry (MS/MS) under fast atom bombardment (FAB) conditions. Based on the analysis of the collision-induced fragment-ion spectrum of the single charged molecular ions of both native and partially hydrolyzed forms of lipopeptide, a new general structure of lichenysin A components was elucidated. It varies from previously proposed structure by having in the peptide portion of lipopeptide the L-Gln-1 and L-Asp-5 residues instead of L-Glu-1 and L-Asn-5. The verified chemical structure of lichenysin A was found to be reflected in the structural organization of the corresponding lichenysin A synthetase, LchA, described recently. ß 1999 Published by Elsevier Science B.V. All rights reserved.
1. Introduction Lichenysin A is a complex of cyclic lipoheptapeptides produced by several Bacillus licheniformis strains and exhibits a broad range inhibitory activity against both Gram-negative and Gram-positive bacteria [1]. Moreover, lichenysin A reduces the surface tension of water from 72 to 28 mN/m with a critical micelle concentration as little as 12 WM, comparing
* Corresponding author. CSRAFA Sicilian Center for Atmospheric Research and Environmental Physics c/o Dipartamento di Fisica della Materia, Universita¨ di Messina, Salina Sperone 31, 98166 Messina, Italy. Fax: +39-90-676-5505; E-mail: [email protected]
favorably with other known powerful surface-active agents [1]. Biosurfactants are of increasing interest due to their broad range of potential applications in the cosmetic, food, health care, pulp- and paperprocessing, coal, ceramic, and metal industries [2]. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants [3^5]. Towards the large-scale industrial production of biosurfactants, the physiology, biochemistry and genetics of biosurfactant synthesis has to be well understood. The detailed characterization of lichenysin A complex showed that this is a mixture of at least 14 components ranging in size from 992.0 to 1034.0
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Da and varying in their lipophilic moiety composed of n-, iso- and anteiso-L-hydroxy fatty acids of di¡erent length. Addition of branched-chain K-amino acids to the medium caused the similar changes in lipophilic moieties of lichenysin A and decrease surface activity of the [6]. Recently, the molecular biology of lichenysin A biosynthesis have been partially clari¢ed. The structural and at least one of the regulatory genes encoding the lichenysin A biosynthesis pathway have been isolated and characterized [7,8]. As many bioactive peptides, lichenysin A is synthetased non-ribosomally on a large peptide synthetase using a thiotemplate mechanism. Lichenysin A synthetase, LchA, as well as other peptide-, fatty acidand polyketide synthetases is representative of large multienzyme system with modular organization. It has been demonstrated that arrangement of the modules (1000^1500 amino acid residues) along the multifunctional polypeptide chain de¢nes the nature and sequence of the amino acid components in peptide product [9]. Analyzing the structural architecture of LchA synthetase which contains seven amino acid activation^thiolation, two epimerization and one thioesterase modules, we assumed the new chemical structure of lichenysin A, slightly di¡erent from we proposed previously. This paper describes the structural veri¢cation of abundant lichenysin A components using FAB-MS/MS analysis. 2. Materials and methods The lichenysin A producing strains of B. licheniformis BNP29 and BAS50 isolated from a North German oil reservoir at a depth of 1500 m [10] and surfactin-producer Bacillus subtilis ATCC 21332 were used in this study. The basic medium for cultivation of all strains was modi¢ed Copper's minimal medium (CMM), supplemented with 2% (w/v) glucose [1]. Bacterial growth in CMM in 2-l shake-£asks at 30³C was monitored by measuring the optical density at 600 nm. The surface tension of spent medium was determined with a ring tensiometer (K6; Kru«ss, Hamburg, Germany). At T7 and T27 of the growth curve, respectively, when surfactant production was known to reach its maximum, the surface-active cyclic lipopeptides lichenysin A and surfactin were removed from the medium as
described previously [1,7,11]. The crude material obtained after acid precipitation was further puri¢ed by column chromatography (BpJ Inert SPE, Silica, Burdick and Jackson, USA). The columns were conditioned by overnight heating at 100³C and, after cooling to room temperature, with 10 ml of dichloromethane. The surfactants were eluted from the column with solvents of gradually increasing polarity [1], dried under nitrogen and further puri¢ed by thin-layer chromatography (TLC) on 1-mm silicacoated glass plates (20U20 cm) (Merck, Darmstadt, Germany) in solvent system CHCl3 /CH3 OH/28% NH4 OH, 65:25:4, v/v/v. The lichenysin A and surfactin found at Rf 0.45 and 0.20, respectively, were used for mass spectrometric measurements as native and partially hydrolyzed forms. A mild alkaline hydrolysis of the cyclic lipopeptides with 1 M NaOH at 40³C for 24 h was used to obtain the partially hydrolyzed fragments of the surfactants. 2.1. Enantiospeci¢c analysis of lichenysin A-constituent amino acids Native lipopeptide was hydrolyzed in 6 M HCl at 110³C for 24 h in sealed tubes and methanol-soluble products were dried after repeated evaporation with ethanol. The amino acids were separated on silicacoated TLC plates (Merck, Darmstadt, Germany) in solvent system C4 H9 OH/CH3 COCH3 /H2 O, 32:48:8, v/v/v, scraped o¡, extracted with CHCl3 /CH3 OH (2:1, v/v) and supplied on chiral CHIR 25 TLC plates (Merck, Darmstadt, Germany) along with standard amino acids in both L- and D-con¢guration. The solvent system used was CH3 OH/CH3 CN/H2 O (12:40:8, v/v/v). Amino acid stains were visualized by plate spraying with ninhydrin (2% solution in acetone). 2.2. Fast atom bombardment mass spectrometry (FAB-MS) FAB-MS in the positive and negative mode was performed on the ¢rst of two spectrometers of a tandem high-resolution instrument of E1 B1 E2 B2 con¢guration (JMS-HX/HX110A, Jeol, Tokyo, Japan) at 10 kV accelerating voltage. The MS resolution was set to 1:1500. The Jeol FAB gun was operated at 6 kV with xenon as the FAB gas. A mixture of
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triethanolamine and tetramethylurea (Japanese matrix) was used as a matrix for lipopeptide analysis. 2.3. Tandem mass spectrometry (FAB-MS/MS) Both negative and positive daughter ion spectra were recorded using all four sectors of the tandem mass spectrometer. The precursor ions were decomposed at the collision cell under high energy collision-induced dissociation (CID) which took place in the third ¢eld free region. Helium served as the collision gas at a pressure su¤cient to reduce the precursor ion signal to 30% of the original value. The collision cell was operated at 3 kV or at ground potential in the positive and at ground potential in the negative mode. Resolution of the product ion spectra (MS2) was set to 1:1000. FAB-CID spectra (linked scans of MS2 at constant B/E ratio) were obtained by processing the mass data using a JEOL JMA-DA7000 data system. 3. Results B. licheniformis and B. subtilis strains were harvested in the middle logarithmic and in the late stationary phase of growth, respectively, corresponding to the maximum rate of surface-active lipopeptide production. The products were isolated and their purity checked by TLC. To verify the absolute structure
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of lichenysin A, especially the position of amino acid amide, the surfactant was re-examined by using direct tandem mass spectrometry analysis of its native and partially hydrolyzed forms. Lichenysin A, as it was proposed before by NMR analysis and (+)-FAB-MS/MS analysis of O,N-permethylated [1], has seven, invariant amino acid residues in its peptide moiety with the following sequence: Glx-Leu-Leu-Val-Asx-Leu-Ile, and is a mixture of isomeric and homologous compounds differing by the lipid parts representing by 14 linear and branched L-hydroxy fatty acids ranging in size from C12 to C17 . In the positive FAB ionization MS mode, the most abundant molecular ions [M+H] were detected at m/z 1035, 1021 and 1007 [1]. The negative FAB ionization MS of native lichenysin A showed deprotonated molecular ions [M3H]3 at m/z 1061, 1047, 1033, 1019, 1005 and 991 with m/z 1033, 1019 and 1005 being the major ions (Fig. 1). Such a distribution of molecular ions is in excellent agreement with data obtained by fatty acid analysis of lichenysin A mixture. The main L-hydroxy fatty acid residues in the lipophilic part of lichenysin A molecules was shown to be C13 , C14 and C15 acids, 7.3, 29.9 and 59.3%, respectively [1]. The absolute con¢guration of the individual constituent amino acids was established by using total acidic hydrolysis and the obtained amino acids after extraction were analyzed on chiral plates. The results shown on Fig. 2 clearly demonstrated that only leu-
Fig. 1. Partial (3)-FAB spectrum of the native lichenysin A fraction.
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Fig. 2. Thin layer chromatography on CHIR 25 TLC plates of previously separated lichenysin A-constituent amino acids. S, corresponding standard amino acid in both con¢guration (bolded in white); H, amino acid obtained after hydrolysis of lichenysin A molecule.
cine is present in peptide moiety of lichenysin A in both L- and D-forms. Tandem mass spectrometry (MS/MS) is ideally suited for the establishing the structure of the major components in the mixture of structurally closed compounds without their puri¢cation. We previously applied MS/MS to the structural characterization of novel glycine-containing glucolipids from the hydrocarbonoclastic, biosurfactant-producing marine microorganism Alcanivorax borkumensis under FAB conditions, and satisfactory product ion spectra containing many sequence ions were obtained [12]. To con¢rm the proposed structures of lichenysin A-related components, especially their amino acid composition and the exact position of amino acid amide in the peptide moiety of lipopeptide, the isolated compounds were analyzed by MS/MS and the fragment ions in the product ion spectra were examined in detail and compared with that of surfactin, the surface-active lipopeptide from B. subtilis, structurally similar to lichenysin A and the amino acid sequence of which is known [13]. To obtain the sequence ions of the investigated surface-active lipopeptides with di¡erent molecular masses, the singly charged ions were collision activated. Positive FAB collision-induced decomposition (CID) of the surfactin molecular ion species resulted in the production of many structurally informative fragments, such as immonium ions originating from constituent amino acid, N-terminal (an ) and C-terminal (xn ) ions. The resulting tandem MS spectrum of the surfactin ion at m/z 1022 which corresponds to N-(3-hydroxytetrade-
canoyl) surfactin, C14 -surfactin, is shown in Fig. 3A. Fragment ions a7 at m/z 909 was detected which was attributed to the neutral loss of 113 amu (loss of dehydrated leucine). The consequential loss of the masses m/z a7 , 909Ca6 , 796 (909^113)Ca5 , 681 (796^115)Ca4 , 582 (681^99)Ca3 , 469 (582^ 113)Ca2 , 356 (469^113)Ca1 , 227 (365^129) which corresponds to the following loss of the N-terminal amino acid residues of surfactin molecule, was detected. Only several C-terminal ions were identi¢ed in the FAB CID MS spectrum, namely x4 and x5 , at m/z 441 and 554, which were attributed to the H adducts of cleaved o¡ tetra- and pentapeptide, respectively. Similar fragmentation patterns were observed in the positive FAB CID MS spectrum of molecular ions of lichenysin A (Fig. 3B,C). The collision activated singly charged ion at m/z 1021, corresponding to C14 -lichenysin A, gave the fragmentation sequences at m/z a7 , 908Ca6 , 795Ca5 , 680Ca4 , 581Ca3 , 468Ca2 , 355Ca1 , 227. The main C-terminal ions x3 and x4 , at m/z 441 and 554 were also detectable for this molecular ion of lichenysin A. The one proton di¡erence obtained for the a7 ^a2 ions in MS spectra of surfactin and lichenysin A could be explain only by the presence of glutamine as the N-terminal amino acid in the peptide moiety of lichenysin A instead of glutamate of surfactin. Taking into consideration the possible fragmentation of both C14 -surfactin and C14 -lichenysin A, the common for both lipopeptides fragment ion a1 at m/z 227 could be interpreted also as a protonated x2 ion. This ion, in fact, is replaced by m/z
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Fig. 3. Positive FAB-CID spectrum of C14 -surfactin, m/z 1022 (A), C14 -lichenysin A, m/z 1021 (B) and C15 -lichenysin A, m/z 1035 (C). For a discussion of the observed daughter ions, see text and Fig. 4.
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Fig. 4. Cleavage sites and fragmentation mode of two proposed isomeric dehydrated species of opened form of C14 -lichenysin A as derived from singly charged N- and C-terminal fragment signals obtained from the (+)-FAB-CID-MS experiments.
241 in the positive FAB CID MS spectrum of molecular ions of lichenysin A at m/z 1035, which corresponds to the C15 -lichenysin A, suggesting its attribution to the hydrogenated L-hydroxy-pentadecanoic acid replacing L-hydroxy-tetradecanoic acid (Fig. 3C). Besides xn and an series of fragmentation sequence, several other fragments belonging to the di¡erent series were observed. Such mode of fragmentation could readily be explained by the presence of the molecular ions of two isomeric species of opened form, respectively dehydrated at C-terminal isoleucine or at the L-hydroxy group of fatty acid. In fact, we could not detect the presence of the doubly charged molecular ions [M32H]23 or [M+2H]2 in negative or positive FAB mode, respectively. Taking into consideration the cyclic form of native lichenysin A due to lactone linkage between L-hydroxy group of fatty acid and carboxy group of C-terminal isoleucine, this ¢nding could be explained by the spontaneous opening of the native form of lichenysin A under FAB conditions followed by simultaneous formation of two isomeric dehydrated species of opened form (Fig. 4). The yn and bn series, which
Fig. 5. Positive FAB-CID fragment-ion spectrum of the singly charged mild hydrolyzed product-ion at m/z 685. Proposed structure and fragment-ions are shown by indicating the m/z values of the corresponding main peaks.
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MS/MS results, we propose a new structure of lichenysin A di¡ering from the previously published structure by the presence of Asp-5 and Gln-1 as N-terminal amino acid in the peptide part of the investigated surfactant. 4. Discussion
Fig. 6. General structure of lichenysin A components.
belong to the fragmentation sequence of opened C14 and C15 -lichenysin A dehydrated at the C3 -position of fatty acid, is characterized, respectively, by the presence of abundant molecular ions, y6 and b2 , at m/z 685 and 337 and at m/z 685 and 351 (Figs. 3B,C and 4). A similar mode of fragmentation was detected in the FAB-CID spectrum of molecular ion of surfactin at m/z 1022 (Fig. 3A), and the ions at m/z 685 and 338 could be characterized as the main compounds. Because the lichenysin A components, especially C14 -lichenysin A, give, in several cases, the same Cand N-terminal fragmentation patterns, complexing the interpretation of FAB-CID spectrum, the ion spectra of their mild hydrolyzed product were examined in detail for sequence ions. The positive FABMS analysis revealed the presence of abundant molecular ion [M+H] at m/z 685. This mass is appreciably lower than 812.0 Da, the mass calculated for the free heptapeptide derived from the native lichenysin A. The mass di¡erence of 128.0 corresponds exactly to the free hexapeptide structure obtained after cleavages at Leu-2 and Ile-7. To prove this hypothesis and to obtain the sequence of the investigated peptide, the singly charged ion at m/z 685 was collision activated. As shown in Fig. 5, all of the expected C-terminal and N-terminal sequence ions and also immonium ions originating from constituent amino acid were observed. Thus, based on the
The structure and composition of the cyclic lipopeptide surfactant, lichenysin A have been studied by a variety of analytical techniques. Following these results, we assumed that lichenysin A is a mixture of structurally similar components with invariant peptide polar head and di¡erent hydrophobic tales representing by 14 L-hydroxy C12 ^C17 fatty acid residues [1,6]. Based on the amino acid analysis, NMR data and MS/MS spectrum of permethylated form of lichenysin A, the peptide moiety was found to contain seven amino acid residues pro molecule of surfactant with the following sequence: Glx-Leu-LeuVal-Asx-Leu-Ile. The abundant structural analogs of lichenysin A range in even-numbered molecular weights from 1006 to 1034 Da, while the structurally similar surfactin has odd-numbered molecular weights in the range of 1007^1035 Da. Taking this ¢nding into consideration together with the fact that lichenysin A is less polar than surfactin, we assumed the presence of one amino acid amide within the peptide moiety either at the ¢rst or at the ¢fth position. Based on the results obtained after the reduction with LiBH4 made to clarify the exact position of amino acid amide, the structure of lichenysin A having Asn-5 was proposed [1]. In our recent work, we showed the modular organization of lichenysin A synthetase LchA. The lchAA gene product (LchAA) contains three modules, with a C-terminal epimerization domain attached to the third; lchAB encodes LchAB, and has similar structure to LchAA; lchAC encodes LchAC, one module with an additional carboxy-terminal putative thioesterase domain [8]. Since the chemical structure of produced peptides is re£ected in the gene and protein sequences of corresponding synthetases [9,14], the multi-modular LchA, composed from seven modules, should consequentially incorporate seven amino acids during the peptide synthesis. The third and sixth modules of LchAA
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and LchAB, respectively, contain at their C-terminal ends motifs found in all synthetases producing Damino acid-containing peptides and known as epimerization domains [14^17]. We con¢rmed the presence of D-Leu residues in molecule of lichenysin A by TLC-checking of the absolute con¢guration of the individual constituent amino acids on chiral plates (Fig. 2). This ¢nding, taken together with the occurrence of two epimerization domains respectively coupled with LchAA3 and LchAB3 modules of lichenysin A synthetase [8], suggests the presence in the lichenysin A molecule at position 3 and 6 of two amino acid residues in D-con¢guration, namely Leu-3 and Leu-6. Recent studies on amino acid-activating modules of peptide synthetases have shown that these modules harbor the speci¢city pockets for binding of the amino acid substrate and thereby dictate the primary structure of the ¢nal peptide [14,18]. The comparative sequence analysis of the substrate-recognizing pockets of LchA synthetase with those of known synthetases recognizing the same amino acids revealed that the LchA pocket which binds the ¢fth amino acid was found to be identical to that of Asp-binding pockets of surfactin and bacitracin synthetases, while the structural organization of the substrate-recognizing pocket accommodating the ¢rst amino acid is more similar to the Gln-binding pocket of tyrocidine synthetase [8]. Based on these results, we assumed a novel general structure of lichenysin A components, where the L-Gln-1 and L-Asp-5 residues are present in the peptide portion of lipopeptide instead of L-Glu-1 and L-Asn-5, proposed previously [1]. To con¢rm this hypothesis, the structural veri¢cation of peptide polar head of lichenysin A was made by tandem mass spectrometry analysis of its native and partially hydrolyzed forms. The abundant structural analogs of lichenysin A were the same as that proposed previously except that N-terminal L-glutamate and L-asparagine-5 were replaced by L-glutamine and L-aspartate, respectively (Fig. 6). It was con¢rmed that the main lichenysin A components with molecular masses Mr of 1006, 1020 and 1034 Da contain L-hydroxy-tridecanoic, L-hydroxy-tetradecanoic and L-hydroxy-pentadecanoic acids, respectively. Minor compounds with shorter or longer fatty acids were also detected.
Acknowledgements We are grateful to Prof. Dr. K.N. Timmis, Director of GBF-Division of Microbiology and Prof. Dr. V. Grasso for their encouragement and for helpful discussions. We are indebted to P. Wolf and I. Grammel for their excellent technical assistance. This work was supported by a fund of the German Federal Ministry for Science, Education and Research (Project 0319433C). References [1] M.M. Yakimov, K.N. Timmis, V. Wray, H.L. Fredrickson, Appl. Environ. Microbiol. 61 (1995) 1706^1713. [2] J.D. Desai, I.M. Banat, Microbiol. Mol. Biol. Rev. 61 (1997) 47^64. [3] K. Scheibenbogen, R.G. Zytner, H. Lee, J.T. Trevors, J. Chem. Tech. Biotechnol. 59 (1994) 53^59. [4] M.A. Providenti, C.A. Flemming, H. Lee, J.T. Trevors, FEMS Microbiol. Ecol. 17 (1995) 15^26. [5] M.M. Yakimov, M.M. Amro, M. Bock, K. Boeseker, H.L. Fredrickson, D.G. Kessel, K.N. Timmis, J. Petrol. Sci. Engineer. 18 (1997) 146^162. [6] M.M. Yakimov, H.L. Fredrickson, K.N. Timmis, Biotechn. Appl. Biochem. 23 (1996) 13^18. [7] M.M. Yakimov, P.N. Golyshin, Biotechnol. Prog. 13 (1997) 757^761. [8] M.M. Yakimov, A. Kroeger, T.N. Slepak, L. Giuliano, K.N. Timmis, P.N. Golyshin, Biochim. Biophys. Acta 1399 (1998) 141^153. [9] J. Vater, T. Stein, D. Vollenbroich, V. Kruft, B. WittmannLiebold, P. Franke, L. Liu, P. Zuber, J. Protein Chem. 16 (1997) 557^564. [10] M. Bock, P. Ka«mpfer, K. Bosecker, W. Dott, Appl. Microbiol. Biotechnol. 42 (1994) 463^468. [11] S.C. Lin, M.M. Sharma, G. Georgiou, Biotechnol. Prog. 9 (1993) 138^145. [12] W.-R. Abraham, H. Meyer, M. Yakimov, Biochim. Biophys. Acta 1393 (1998) 57^62. [13] K. Arima, A. Kakinuma, G. Tamura, Biochem. Biophys. Res. Commun. 31 (1968) 488. [14] M.A. Marahiel, Chem. Biol. 4 (1997) 561^567. [15] H.D. Mootz, M.A. Marahiel, J. Bacteriol. 179 (1997) 6843^ 6850. [16] D. Konz, A. Klens, K. Schorgendorfer, M.A. Marahiel, Chem. Biol. 4 (1997) 927^937. [17] K. Turgay, M. Krause, M.A. Marahiel, Mol. Microbiol. 6 (1992) 529^546. [18] E. Conti, T. Stachelhaus, M.A. Marahiel, P. Brink, EMBO J. 16 (1997) 4174^4183.
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Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry Michail M. Yakimov a
a; b;
*, Wolf-Rainer Abraham a , Holger Meyer a , Laura Giuliano c , Peter N. Golyshin a
Department of Microbiology, GBF National Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany b CSRAFA Sicilian Center for Atmospheric Research and Environmental Physics c/o Dipartamento di Fisica della Materia, Universita¨ di Messina, Salina Sperone 31, 98166 Messina, Italy c Dipartamento di Biologia Animale ed Ecologia Marina, Universita' di Messina, Salina Sperone 31, 98166 Messina, Italy Received 30 November 1998; received in revised form 18 March 1999; accepted 24 March 1999
Abstract The structural characterization of the cyclic lipoheptapeptide surfactant lichenysin A components, produced by Bacillus licheniformis strains via the non-ribosomal pathway on a corresponding peptide synthetase, was carried out using a tandem mass spectrometry (MS/MS) under fast atom bombardment (FAB) conditions. Based on the analysis of the collision-induced fragment-ion spectrum of the single charged molecular ions of both native and partially hydrolyzed forms of lipopeptide, a new general structure of lichenysin A components was elucidated. It varies from previously proposed structure by having in the peptide portion of lipopeptide the L-Gln-1 and L-Asp-5 residues instead of L-Glu-1 and L-Asn-5. The verified chemical structure of lichenysin A was found to be reflected in the structural organization of the corresponding lichenysin A synthetase, LchA, described recently. ß 1999 Published by Elsevier Science B.V. All rights reserved.
1. Introduction Lichenysin A is a complex of cyclic lipoheptapeptides produced by several Bacillus licheniformis strains and exhibits a broad range inhibitory activity against both Gram-negative and Gram-positive bacteria [1]. Moreover, lichenysin A reduces the surface tension of water from 72 to 28 mN/m with a critical micelle concentration as little as 12 WM, comparing
* Corresponding author. CSRAFA Sicilian Center for Atmospheric Research and Environmental Physics c/o Dipartamento di Fisica della Materia, Universita¨ di Messina, Salina Sperone 31, 98166 Messina, Italy. Fax: +39-90-676-5505; E-mail: [email protected]
favorably with other known powerful surface-active agents [1]. Biosurfactants are of increasing interest due to their broad range of potential applications in the cosmetic, food, health care, pulp- and paperprocessing, coal, ceramic, and metal industries [2]. However, the most promising applications are cleaning of oil-contaminated tankers, oil spill management, transportation of heavy crude oil, enhanced oil recovery, recovery of crude oil from sludge, and bioremediation of sites contaminated with hydrocarbons, heavy metals, and other pollutants [3^5]. Towards the large-scale industrial production of biosurfactants, the physiology, biochemistry and genetics of biosurfactant synthesis has to be well understood. The detailed characterization of lichenysin A complex showed that this is a mixture of at least 14 components ranging in size from 992.0 to 1034.0
1388-1981 / 99 / $ ^ see front matter ß 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 0 5 8 - X
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Da and varying in their lipophilic moiety composed of n-, iso- and anteiso-L-hydroxy fatty acids of di¡erent length. Addition of branched-chain K-amino acids to the medium caused the similar changes in lipophilic moieties of lichenysin A and decrease surface activity of the [6]. Recently, the molecular biology of lichenysin A biosynthesis have been partially clari¢ed. The structural and at least one of the regulatory genes encoding the lichenysin A biosynthesis pathway have been isolated and characterized [7,8]. As many bioactive peptides, lichenysin A is synthetased non-ribosomally on a large peptide synthetase using a thiotemplate mechanism. Lichenysin A synthetase, LchA, as well as other peptide-, fatty acidand polyketide synthetases is representative of large multienzyme system with modular organization. It has been demonstrated that arrangement of the modules (1000^1500 amino acid residues) along the multifunctional polypeptide chain de¢nes the nature and sequence of the amino acid components in peptide product [9]. Analyzing the structural architecture of LchA synthetase which contains seven amino acid activation^thiolation, two epimerization and one thioesterase modules, we assumed the new chemical structure of lichenysin A, slightly di¡erent from we proposed previously. This paper describes the structural veri¢cation of abundant lichenysin A components using FAB-MS/MS analysis. 2. Materials and methods The lichenysin A producing strains of B. licheniformis BNP29 and BAS50 isolated from a North German oil reservoir at a depth of 1500 m [10] and surfactin-producer Bacillus subtilis ATCC 21332 were used in this study. The basic medium for cultivation of all strains was modi¢ed Copper's minimal medium (CMM), supplemented with 2% (w/v) glucose [1]. Bacterial growth in CMM in 2-l shake-£asks at 30³C was monitored by measuring the optical density at 600 nm. The surface tension of spent medium was determined with a ring tensiometer (K6; Kru«ss, Hamburg, Germany). At T7 and T27 of the growth curve, respectively, when surfactant production was known to reach its maximum, the surface-active cyclic lipopeptides lichenysin A and surfactin were removed from the medium as
described previously [1,7,11]. The crude material obtained after acid precipitation was further puri¢ed by column chromatography (BpJ Inert SPE, Silica, Burdick and Jackson, USA). The columns were conditioned by overnight heating at 100³C and, after cooling to room temperature, with 10 ml of dichloromethane. The surfactants were eluted from the column with solvents of gradually increasing polarity [1], dried under nitrogen and further puri¢ed by thin-layer chromatography (TLC) on 1-mm silicacoated glass plates (20U20 cm) (Merck, Darmstadt, Germany) in solvent system CHCl3 /CH3 OH/28% NH4 OH, 65:25:4, v/v/v. The lichenysin A and surfactin found at Rf 0.45 and 0.20, respectively, were used for mass spectrometric measurements as native and partially hydrolyzed forms. A mild alkaline hydrolysis of the cyclic lipopeptides with 1 M NaOH at 40³C for 24 h was used to obtain the partially hydrolyzed fragments of the surfactants. 2.1. Enantiospeci¢c analysis of lichenysin A-constituent amino acids Native lipopeptide was hydrolyzed in 6 M HCl at 110³C for 24 h in sealed tubes and methanol-soluble products were dried after repeated evaporation with ethanol. The amino acids were separated on silicacoated TLC plates (Merck, Darmstadt, Germany) in solvent system C4 H9 OH/CH3 COCH3 /H2 O, 32:48:8, v/v/v, scraped o¡, extracted with CHCl3 /CH3 OH (2:1, v/v) and supplied on chiral CHIR 25 TLC plates (Merck, Darmstadt, Germany) along with standard amino acids in both L- and D-con¢guration. The solvent system used was CH3 OH/CH3 CN/H2 O (12:40:8, v/v/v). Amino acid stains were visualized by plate spraying with ninhydrin (2% solution in acetone). 2.2. Fast atom bombardment mass spectrometry (FAB-MS) FAB-MS in the positive and negative mode was performed on the ¢rst of two spectrometers of a tandem high-resolution instrument of E1 B1 E2 B2 con¢guration (JMS-HX/HX110A, Jeol, Tokyo, Japan) at 10 kV accelerating voltage. The MS resolution was set to 1:1500. The Jeol FAB gun was operated at 6 kV with xenon as the FAB gas. A mixture of
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triethanolamine and tetramethylurea (Japanese matrix) was used as a matrix for lipopeptide analysis. 2.3. Tandem mass spectrometry (FAB-MS/MS) Both negative and positive daughter ion spectra were recorded using all four sectors of the tandem mass spectrometer. The precursor ions were decomposed at the collision cell under high energy collision-induced dissociation (CID) which took place in the third ¢eld free region. Helium served as the collision gas at a pressure su¤cient to reduce the precursor ion signal to 30% of the original value. The collision cell was operated at 3 kV or at ground potential in the positive and at ground potential in the negative mode. Resolution of the product ion spectra (MS2) was set to 1:1000. FAB-CID spectra (linked scans of MS2 at constant B/E ratio) were obtained by processing the mass data using a JEOL JMA-DA7000 data system. 3. Results B. licheniformis and B. subtilis strains were harvested in the middle logarithmic and in the late stationary phase of growth, respectively, corresponding to the maximum rate of surface-active lipopeptide production. The products were isolated and their purity checked by TLC. To verify the absolute structure
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of lichenysin A, especially the position of amino acid amide, the surfactant was re-examined by using direct tandem mass spectrometry analysis of its native and partially hydrolyzed forms. Lichenysin A, as it was proposed before by NMR analysis and (+)-FAB-MS/MS analysis of O,N-permethylated [1], has seven, invariant amino acid residues in its peptide moiety with the following sequence: Glx-Leu-Leu-Val-Asx-Leu-Ile, and is a mixture of isomeric and homologous compounds differing by the lipid parts representing by 14 linear and branched L-hydroxy fatty acids ranging in size from C12 to C17 . In the positive FAB ionization MS mode, the most abundant molecular ions [M+H] were detected at m/z 1035, 1021 and 1007 [1]. The negative FAB ionization MS of native lichenysin A showed deprotonated molecular ions [M3H]3 at m/z 1061, 1047, 1033, 1019, 1005 and 991 with m/z 1033, 1019 and 1005 being the major ions (Fig. 1). Such a distribution of molecular ions is in excellent agreement with data obtained by fatty acid analysis of lichenysin A mixture. The main L-hydroxy fatty acid residues in the lipophilic part of lichenysin A molecules was shown to be C13 , C14 and C15 acids, 7.3, 29.9 and 59.3%, respectively [1]. The absolute con¢guration of the individual constituent amino acids was established by using total acidic hydrolysis and the obtained amino acids after extraction were analyzed on chiral plates. The results shown on Fig. 2 clearly demonstrated that only leu-
Fig. 1. Partial (3)-FAB spectrum of the native lichenysin A fraction.
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Fig. 2. Thin layer chromatography on CHIR 25 TLC plates of previously separated lichenysin A-constituent amino acids. S, corresponding standard amino acid in both con¢guration (bolded in white); H, amino acid obtained after hydrolysis of lichenysin A molecule.
cine is present in peptide moiety of lichenysin A in both L- and D-forms. Tandem mass spectrometry (MS/MS) is ideally suited for the establishing the structure of the major components in the mixture of structurally closed compounds without their puri¢cation. We previously applied MS/MS to the structural characterization of novel glycine-containing glucolipids from the hydrocarbonoclastic, biosurfactant-producing marine microorganism Alcanivorax borkumensis under FAB conditions, and satisfactory product ion spectra containing many sequence ions were obtained [12]. To con¢rm the proposed structures of lichenysin A-related components, especially their amino acid composition and the exact position of amino acid amide in the peptide moiety of lipopeptide, the isolated compounds were analyzed by MS/MS and the fragment ions in the product ion spectra were examined in detail and compared with that of surfactin, the surface-active lipopeptide from B. subtilis, structurally similar to lichenysin A and the amino acid sequence of which is known [13]. To obtain the sequence ions of the investigated surface-active lipopeptides with di¡erent molecular masses, the singly charged ions were collision activated. Positive FAB collision-induced decomposition (CID) of the surfactin molecular ion species resulted in the production of many structurally informative fragments, such as immonium ions originating from constituent amino acid, N-terminal (an ) and C-terminal (xn ) ions. The resulting tandem MS spectrum of the surfactin ion at m/z 1022 which corresponds to N-(3-hydroxytetrade-
canoyl) surfactin, C14 -surfactin, is shown in Fig. 3A. Fragment ions a7 at m/z 909 was detected which was attributed to the neutral loss of 113 amu (loss of dehydrated leucine). The consequential loss of the masses m/z a7 , 909Ca6 , 796 (909^113)Ca5 , 681 (796^115)Ca4 , 582 (681^99)Ca3 , 469 (582^ 113)Ca2 , 356 (469^113)Ca1 , 227 (365^129) which corresponds to the following loss of the N-terminal amino acid residues of surfactin molecule, was detected. Only several C-terminal ions were identi¢ed in the FAB CID MS spectrum, namely x4 and x5 , at m/z 441 and 554, which were attributed to the H adducts of cleaved o¡ tetra- and pentapeptide, respectively. Similar fragmentation patterns were observed in the positive FAB CID MS spectrum of molecular ions of lichenysin A (Fig. 3B,C). The collision activated singly charged ion at m/z 1021, corresponding to C14 -lichenysin A, gave the fragmentation sequences at m/z a7 , 908Ca6 , 795Ca5 , 680Ca4 , 581Ca3 , 468Ca2 , 355Ca1 , 227. The main C-terminal ions x3 and x4 , at m/z 441 and 554 were also detectable for this molecular ion of lichenysin A. The one proton di¡erence obtained for the a7 ^a2 ions in MS spectra of surfactin and lichenysin A could be explain only by the presence of glutamine as the N-terminal amino acid in the peptide moiety of lichenysin A instead of glutamate of surfactin. Taking into consideration the possible fragmentation of both C14 -surfactin and C14 -lichenysin A, the common for both lipopeptides fragment ion a1 at m/z 227 could be interpreted also as a protonated x2 ion. This ion, in fact, is replaced by m/z
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Fig. 3. Positive FAB-CID spectrum of C14 -surfactin, m/z 1022 (A), C14 -lichenysin A, m/z 1021 (B) and C15 -lichenysin A, m/z 1035 (C). For a discussion of the observed daughter ions, see text and Fig. 4.
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Fig. 4. Cleavage sites and fragmentation mode of two proposed isomeric dehydrated species of opened form of C14 -lichenysin A as derived from singly charged N- and C-terminal fragment signals obtained from the (+)-FAB-CID-MS experiments.
241 in the positive FAB CID MS spectrum of molecular ions of lichenysin A at m/z 1035, which corresponds to the C15 -lichenysin A, suggesting its attribution to the hydrogenated L-hydroxy-pentadecanoic acid replacing L-hydroxy-tetradecanoic acid (Fig. 3C). Besides xn and an series of fragmentation sequence, several other fragments belonging to the di¡erent series were observed. Such mode of fragmentation could readily be explained by the presence of the molecular ions of two isomeric species of opened form, respectively dehydrated at C-terminal isoleucine or at the L-hydroxy group of fatty acid. In fact, we could not detect the presence of the doubly charged molecular ions [M32H]23 or [M+2H]2 in negative or positive FAB mode, respectively. Taking into consideration the cyclic form of native lichenysin A due to lactone linkage between L-hydroxy group of fatty acid and carboxy group of C-terminal isoleucine, this ¢nding could be explained by the spontaneous opening of the native form of lichenysin A under FAB conditions followed by simultaneous formation of two isomeric dehydrated species of opened form (Fig. 4). The yn and bn series, which
Fig. 5. Positive FAB-CID fragment-ion spectrum of the singly charged mild hydrolyzed product-ion at m/z 685. Proposed structure and fragment-ions are shown by indicating the m/z values of the corresponding main peaks.
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MS/MS results, we propose a new structure of lichenysin A di¡ering from the previously published structure by the presence of Asp-5 and Gln-1 as N-terminal amino acid in the peptide part of the investigated surfactant. 4. Discussion
Fig. 6. General structure of lichenysin A components.
belong to the fragmentation sequence of opened C14 and C15 -lichenysin A dehydrated at the C3 -position of fatty acid, is characterized, respectively, by the presence of abundant molecular ions, y6 and b2 , at m/z 685 and 337 and at m/z 685 and 351 (Figs. 3B,C and 4). A similar mode of fragmentation was detected in the FAB-CID spectrum of molecular ion of surfactin at m/z 1022 (Fig. 3A), and the ions at m/z 685 and 338 could be characterized as the main compounds. Because the lichenysin A components, especially C14 -lichenysin A, give, in several cases, the same Cand N-terminal fragmentation patterns, complexing the interpretation of FAB-CID spectrum, the ion spectra of their mild hydrolyzed product were examined in detail for sequence ions. The positive FABMS analysis revealed the presence of abundant molecular ion [M+H] at m/z 685. This mass is appreciably lower than 812.0 Da, the mass calculated for the free heptapeptide derived from the native lichenysin A. The mass di¡erence of 128.0 corresponds exactly to the free hexapeptide structure obtained after cleavages at Leu-2 and Ile-7. To prove this hypothesis and to obtain the sequence of the investigated peptide, the singly charged ion at m/z 685 was collision activated. As shown in Fig. 5, all of the expected C-terminal and N-terminal sequence ions and also immonium ions originating from constituent amino acid were observed. Thus, based on the
The structure and composition of the cyclic lipopeptide surfactant, lichenysin A have been studied by a variety of analytical techniques. Following these results, we assumed that lichenysin A is a mixture of structurally similar components with invariant peptide polar head and di¡erent hydrophobic tales representing by 14 L-hydroxy C12 ^C17 fatty acid residues [1,6]. Based on the amino acid analysis, NMR data and MS/MS spectrum of permethylated form of lichenysin A, the peptide moiety was found to contain seven amino acid residues pro molecule of surfactant with the following sequence: Glx-Leu-LeuVal-Asx-Leu-Ile. The abundant structural analogs of lichenysin A range in even-numbered molecular weights from 1006 to 1034 Da, while the structurally similar surfactin has odd-numbered molecular weights in the range of 1007^1035 Da. Taking this ¢nding into consideration together with the fact that lichenysin A is less polar than surfactin, we assumed the presence of one amino acid amide within the peptide moiety either at the ¢rst or at the ¢fth position. Based on the results obtained after the reduction with LiBH4 made to clarify the exact position of amino acid amide, the structure of lichenysin A having Asn-5 was proposed [1]. In our recent work, we showed the modular organization of lichenysin A synthetase LchA. The lchAA gene product (LchAA) contains three modules, with a C-terminal epimerization domain attached to the third; lchAB encodes LchAB, and has similar structure to LchAA; lchAC encodes LchAC, one module with an additional carboxy-terminal putative thioesterase domain [8]. Since the chemical structure of produced peptides is re£ected in the gene and protein sequences of corresponding synthetases [9,14], the multi-modular LchA, composed from seven modules, should consequentially incorporate seven amino acids during the peptide synthesis. The third and sixth modules of LchAA
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and LchAB, respectively, contain at their C-terminal ends motifs found in all synthetases producing Damino acid-containing peptides and known as epimerization domains [14^17]. We con¢rmed the presence of D-Leu residues in molecule of lichenysin A by TLC-checking of the absolute con¢guration of the individual constituent amino acids on chiral plates (Fig. 2). This ¢nding, taken together with the occurrence of two epimerization domains respectively coupled with LchAA3 and LchAB3 modules of lichenysin A synthetase [8], suggests the presence in the lichenysin A molecule at position 3 and 6 of two amino acid residues in D-con¢guration, namely Leu-3 and Leu-6. Recent studies on amino acid-activating modules of peptide synthetases have shown that these modules harbor the speci¢city pockets for binding of the amino acid substrate and thereby dictate the primary structure of the ¢nal peptide [14,18]. The comparative sequence analysis of the substrate-recognizing pockets of LchA synthetase with those of known synthetases recognizing the same amino acids revealed that the LchA pocket which binds the ¢fth amino acid was found to be identical to that of Asp-binding pockets of surfactin and bacitracin synthetases, while the structural organization of the substrate-recognizing pocket accommodating the ¢rst amino acid is more similar to the Gln-binding pocket of tyrocidine synthetase [8]. Based on these results, we assumed a novel general structure of lichenysin A components, where the L-Gln-1 and L-Asp-5 residues are present in the peptide portion of lipopeptide instead of L-Glu-1 and L-Asn-5, proposed previously [1]. To con¢rm this hypothesis, the structural veri¢cation of peptide polar head of lichenysin A was made by tandem mass spectrometry analysis of its native and partially hydrolyzed forms. The abundant structural analogs of lichenysin A were the same as that proposed previously except that N-terminal L-glutamate and L-asparagine-5 were replaced by L-glutamine and L-aspartate, respectively (Fig. 6). It was con¢rmed that the main lichenysin A components with molecular masses Mr of 1006, 1020 and 1034 Da contain L-hydroxy-tridecanoic, L-hydroxy-tetradecanoic and L-hydroxy-pentadecanoic acids, respectively. Minor compounds with shorter or longer fatty acids were also detected.
Acknowledgements We are grateful to Prof. Dr. K.N. Timmis, Director of GBF-Division of Microbiology and Prof. Dr. V. Grasso for their encouragement and for helpful discussions. We are indebted to P. Wolf and I. Grammel for their excellent technical assistance. This work was supported by a fund of the German Federal Ministry for Science, Education and Research (Project 0319433C). References [1] M.M. Yakimov, K.N. Timmis, V. Wray, H.L. Fredrickson, Appl. Environ. Microbiol. 61 (1995) 1706^1713. [2] J.D. Desai, I.M. Banat, Microbiol. Mol. Biol. Rev. 61 (1997) 47^64. [3] K. Scheibenbogen, R.G. Zytner, H. Lee, J.T. Trevors, J. Chem. Tech. Biotechnol. 59 (1994) 53^59. [4] M.A. Providenti, C.A. Flemming, H. Lee, J.T. Trevors, FEMS Microbiol. Ecol. 17 (1995) 15^26. [5] M.M. Yakimov, M.M. Amro, M. Bock, K. Boeseker, H.L. Fredrickson, D.G. Kessel, K.N. Timmis, J. Petrol. Sci. Engineer. 18 (1997) 146^162. [6] M.M. Yakimov, H.L. Fredrickson, K.N. Timmis, Biotechn. Appl. Biochem. 23 (1996) 13^18. [7] M.M. Yakimov, P.N. Golyshin, Biotechnol. Prog. 13 (1997) 757^761. [8] M.M. Yakimov, A. Kroeger, T.N. Slepak, L. Giuliano, K.N. Timmis, P.N. Golyshin, Biochim. Biophys. Acta 1399 (1998) 141^153. [9] J. Vater, T. Stein, D. Vollenbroich, V. Kruft, B. WittmannLiebold, P. Franke, L. Liu, P. Zuber, J. Protein Chem. 16 (1997) 557^564. [10] M. Bock, P. Ka«mpfer, K. Bosecker, W. Dott, Appl. Microbiol. Biotechnol. 42 (1994) 463^468. [11] S.C. Lin, M.M. Sharma, G. Georgiou, Biotechnol. Prog. 9 (1993) 138^145. [12] W.-R. Abraham, H. Meyer, M. Yakimov, Biochim. Biophys. Acta 1393 (1998) 57^62. [13] K. Arima, A. Kakinuma, G. Tamura, Biochem. Biophys. Res. Commun. 31 (1968) 488. [14] M.A. Marahiel, Chem. Biol. 4 (1997) 561^567. [15] H.D. Mootz, M.A. Marahiel, J. Bacteriol. 179 (1997) 6843^ 6850. [16] D. Konz, A. Klens, K. Schorgendorfer, M.A. Marahiel, Chem. Biol. 4 (1997) 927^937. [17] K. Turgay, M. Krause, M.A. Marahiel, Mol. Microbiol. 6 (1992) 529^546. [18] E. Conti, T. Stachelhaus, M.A. Marahiel, P. Brink, EMBO J. 16 (1997) 4174^4183.
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