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A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL) synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

Mareš J, Hájek J, Urajová P, Kopecký J, Hrouzek P - PLoS ONE (2014)

Bottom Line: Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase.High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea).Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology AS CR, v.v.i., Department of Phototrophic Microorganisms - ALGATECH, Třeboň, Czech Republic; Biology Centre of AS CR, v.v.i., Institute of Hydrobiology, České Budějovice, Czech Republic; University of South Bohemia, Faculty of Science, Department of Botany, České Budějovice, Czech Republic.

ABSTRACT
A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

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Related in: MedlinePlus

Fragmentation of 4-methyl-Ahdoa and 4-methyl-Ahtea puwainaphycin F and their chlorinated and hydroxylated analogs.The observed fragmentation losses are denoted by the corresponding amino acid residues. The diagnostic fragments common to all puwainaphycin analogs are marked as Fragments 1–6.
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pone-0111904-g005: Fragmentation of 4-methyl-Ahdoa and 4-methyl-Ahtea puwainaphycin F and their chlorinated and hydroxylated analogs.The observed fragmentation losses are denoted by the corresponding amino acid residues. The diagnostic fragments common to all puwainaphycin analogs are marked as Fragments 1–6.

Mentions: The HPLC-HRMS analysis of crude Cylindrospermum alatosporum extract revealed about 30 pseudomolecular ions in the region 1050–1200 Da, which were attributed to putative puwainaphycin analogs. Puwainaphycin F (4-methyl-Ahtea-PUW-F), together with the congener of m/z 1118.6, were found to be dominant based on UV and MS detection, other variants were observed in trace amounts based on their MS signals (Figure 3B). Of these we have selected eight more prominent peaks for purpose of this study. Their exact masses were measured with high precision (0.0–4.9 ppm), enabling calculation of the elemental composition (Table 1). The MS/MS experiments provided product ions corresponding to identical losses of N-methyl asparagine (Δ 128), dehydrated threonine (Δ 83), alanine (Δ 71), asparagine (Δ 114) and dehydrothereonine (Δ 83) in all compounds detected (Figure S1). Furthermore, the fragmentation pattern of the pseudomolecular ions with m/z 1118.6, 1152.6, 1180.6, 1134.6 and 1162.6 showed clear loss of an additional asparagine and thus have the same amino acid composition as 4-methyl-Ahtea-PUW-F (Figure S1A, B). The loss of glutamine was recognized in compounds m/z 1126.6, 1132.6, 1176.6, 1194.6, and 1166.6; thus, their amino acid sequence is identical to puwainaphycin G (4-methyl-Ahtea-PUW-G; Figure S1C, D). Moreover, the fragmentation of all of these compounds led to the formation of the diagnostic fragments m/z 101.0, 186.1, 198.1, 269.2, 281.2, which were identical for all precursor ions and further confirm the identical primary structure of the cyclic part of the molecule. The ion 281.2 corresponded to fragment C12H16N4O4+H+ (Pro-X1-Val-dThr, where X1 corresponds to the 3-amino-2-oxopropanoyl fragment of the 3-amino-2-hydroxy-4-methyl-fatty acid but where the major part of the aliphatic chain was missing. The remaining diagnostic fragments were derived from ion 281.2. These results confirm that non-identical parts of the puwainaphycin variants are situated in the unusual FA aliphatic chain whereas the remaining part of the molecule is identical for all the puwainaphycin analogs, based on the PUW-F/PUW-G structures. Indeed, within the fragmentation spectra of 4-methyl-Ahtea-PUW-F and its analog 1118, the intense fragment ions m/z 535 and 507, corresponding to sequence Pro-X-Val-DhB (with X as modified FA), were recognized (Figure 4). The mass difference of 28.0291 Da corresponded well with –(CH2)2 on the FA chain (2.2 ppm), and this mass difference was noted in all fragments containing the FA residue (Table S4 A–F). The presence of 3-amino-2-hydroxy-4-methyldodecanoic (4-methyl-Ahdoa) instead of 3-amino-2-hydroxy-4-methyltetradecanoic acid (4-methyl-Ahtea) in the congener 1118.6 was further confirmed by NMR measurements (see Table S5). The 2D NMR spectra (Figure S2) of congener 1118.6 were almost identical to the previously measured NMR spectra of 4-methyl-Ahtea-PUW-F [10]. The only non-identical part of the spectrum was noted in the HSQC region set by a 13C shift of 15–34 ppm and a 1H shift of 0.5–1.5 ppm. Although in 4-methyl-Ahtea-PUW-F 1H13C, the HSQC crosspeaks X-CH21 to X-CH29 were identified, in the case of 4-methyl-Ahdoa-PUW-F, the crosspeaks X-CH28 and X-CH29 were clearly missing (see Figure 5). An identical MS/MS fragmentation pattern was also provided by the ion 1132.6 (Figure S1 C, D), a structural analog of 4-methyl-Ahtea-PUW-G; thus, the 4-methyl-Ahdoa-PUW-G was confirmed as the variant of puwainaphycin G with tetradecanoic acid replaced by dodecanoic acid (see Table S4). The 1H13C HSQC crosspeaks unique for Gln4 in 4-methyl-Ahdoa-PUW-G were also identified in the sample; however, the full assignment of this variant was not successful due to overlapping of very similar signals with 4-methyl-Ahdoa-PUW-F and its low concentration (Table S5). Additionally, in some of less intensive molecular ions observed, the fragmentation analysis suggests presence of analogs with shorter (<C12) and longer (>C14) FA chains (data not shown).


A hybrid non-ribosomal peptide/polyketide synthetase containing fatty-acyl ligase (FAAL) synthesizes the β-amino fatty acid lipopeptides puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum.

Mareš J, Hájek J, Urajová P, Kopecký J, Hrouzek P - PLoS ONE (2014)

Fragmentation of 4-methyl-Ahdoa and 4-methyl-Ahtea puwainaphycin F and their chlorinated and hydroxylated analogs.The observed fragmentation losses are denoted by the corresponding amino acid residues. The diagnostic fragments common to all puwainaphycin analogs are marked as Fragments 1–6.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219810&req=5

pone-0111904-g005: Fragmentation of 4-methyl-Ahdoa and 4-methyl-Ahtea puwainaphycin F and their chlorinated and hydroxylated analogs.The observed fragmentation losses are denoted by the corresponding amino acid residues. The diagnostic fragments common to all puwainaphycin analogs are marked as Fragments 1–6.
Mentions: The HPLC-HRMS analysis of crude Cylindrospermum alatosporum extract revealed about 30 pseudomolecular ions in the region 1050–1200 Da, which were attributed to putative puwainaphycin analogs. Puwainaphycin F (4-methyl-Ahtea-PUW-F), together with the congener of m/z 1118.6, were found to be dominant based on UV and MS detection, other variants were observed in trace amounts based on their MS signals (Figure 3B). Of these we have selected eight more prominent peaks for purpose of this study. Their exact masses were measured with high precision (0.0–4.9 ppm), enabling calculation of the elemental composition (Table 1). The MS/MS experiments provided product ions corresponding to identical losses of N-methyl asparagine (Δ 128), dehydrated threonine (Δ 83), alanine (Δ 71), asparagine (Δ 114) and dehydrothereonine (Δ 83) in all compounds detected (Figure S1). Furthermore, the fragmentation pattern of the pseudomolecular ions with m/z 1118.6, 1152.6, 1180.6, 1134.6 and 1162.6 showed clear loss of an additional asparagine and thus have the same amino acid composition as 4-methyl-Ahtea-PUW-F (Figure S1A, B). The loss of glutamine was recognized in compounds m/z 1126.6, 1132.6, 1176.6, 1194.6, and 1166.6; thus, their amino acid sequence is identical to puwainaphycin G (4-methyl-Ahtea-PUW-G; Figure S1C, D). Moreover, the fragmentation of all of these compounds led to the formation of the diagnostic fragments m/z 101.0, 186.1, 198.1, 269.2, 281.2, which were identical for all precursor ions and further confirm the identical primary structure of the cyclic part of the molecule. The ion 281.2 corresponded to fragment C12H16N4O4+H+ (Pro-X1-Val-dThr, where X1 corresponds to the 3-amino-2-oxopropanoyl fragment of the 3-amino-2-hydroxy-4-methyl-fatty acid but where the major part of the aliphatic chain was missing. The remaining diagnostic fragments were derived from ion 281.2. These results confirm that non-identical parts of the puwainaphycin variants are situated in the unusual FA aliphatic chain whereas the remaining part of the molecule is identical for all the puwainaphycin analogs, based on the PUW-F/PUW-G structures. Indeed, within the fragmentation spectra of 4-methyl-Ahtea-PUW-F and its analog 1118, the intense fragment ions m/z 535 and 507, corresponding to sequence Pro-X-Val-DhB (with X as modified FA), were recognized (Figure 4). The mass difference of 28.0291 Da corresponded well with –(CH2)2 on the FA chain (2.2 ppm), and this mass difference was noted in all fragments containing the FA residue (Table S4 A–F). The presence of 3-amino-2-hydroxy-4-methyldodecanoic (4-methyl-Ahdoa) instead of 3-amino-2-hydroxy-4-methyltetradecanoic acid (4-methyl-Ahtea) in the congener 1118.6 was further confirmed by NMR measurements (see Table S5). The 2D NMR spectra (Figure S2) of congener 1118.6 were almost identical to the previously measured NMR spectra of 4-methyl-Ahtea-PUW-F [10]. The only non-identical part of the spectrum was noted in the HSQC region set by a 13C shift of 15–34 ppm and a 1H shift of 0.5–1.5 ppm. Although in 4-methyl-Ahtea-PUW-F 1H13C, the HSQC crosspeaks X-CH21 to X-CH29 were identified, in the case of 4-methyl-Ahdoa-PUW-F, the crosspeaks X-CH28 and X-CH29 were clearly missing (see Figure 5). An identical MS/MS fragmentation pattern was also provided by the ion 1132.6 (Figure S1 C, D), a structural analog of 4-methyl-Ahtea-PUW-G; thus, the 4-methyl-Ahdoa-PUW-G was confirmed as the variant of puwainaphycin G with tetradecanoic acid replaced by dodecanoic acid (see Table S4). The 1H13C HSQC crosspeaks unique for Gln4 in 4-methyl-Ahdoa-PUW-G were also identified in the sample; however, the full assignment of this variant was not successful due to overlapping of very similar signals with 4-methyl-Ahdoa-PUW-F and its low concentration (Table S5). Additionally, in some of less intensive molecular ions observed, the fragmentation analysis suggests presence of analogs with shorter (<C12) and longer (>C14) FA chains (data not shown).

Bottom Line: Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase.High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea).Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology AS CR, v.v.i., Department of Phototrophic Microorganisms - ALGATECH, Třeboň, Czech Republic; Biology Centre of AS CR, v.v.i., Institute of Hydrobiology, České Budějovice, Czech Republic; University of South Bohemia, Faculty of Science, Department of Botany, České Budějovice, Czech Republic.

ABSTRACT
A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a fatty acid residue via fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-amino-2-hydroxy-4-methyl dodecanoic acid (4-methyl-Ahdoa) or 3-amino-2-hydroxy-4-methyl tetradecanoic acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the fatty acyl-AMP ligase and one of the amino acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-amino fatty acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.

Show MeSH
Related in: MedlinePlus