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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

Proposed biosynthesis of puwainaphycins.A, adenylation domain; ACP, acyl carrier protein; AmT, aminotransferase; AT, acyltransferase; C, condensation domain; DH, dehydratase; E, epimerase; ER, enoylreductase; FAAL, fatty acyl-AMP ligase; MT, methyltransferase; NRPS, non-ribosomal peptide synthetase; KR, ketoreductase; KS, ketosynthetase; Ox, monooxygenase; PCP, peptidyl carrier protein; PKS, polyketide synthetase; TE, thioesterase.
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pone-0111904-g002: Proposed biosynthesis of puwainaphycins.A, adenylation domain; ACP, acyl carrier protein; AmT, aminotransferase; AT, acyltransferase; C, condensation domain; DH, dehydratase; E, epimerase; ER, enoylreductase; FAAL, fatty acyl-AMP ligase; MT, methyltransferase; NRPS, non-ribosomal peptide synthetase; KR, ketoreductase; KS, ketosynthetase; Ox, monooxygenase; PCP, peptidyl carrier protein; PKS, polyketide synthetase; TE, thioesterase.

Mentions: The reconstructed course of puwainaphycin biosynthesis, as visualized in Figure 2, starts with the puwC gene product. This protein contains conserved catalytic domains typical for fatty acyl-AMP ligases (FAALs), enzymes that activate a fatty acyl by adenylation to subsequently serve as a substrate for polyketide synthetases. PuwC also showed a relatively high amino acid sequence identity (approximately 60%) to several annotated cyanobacterial fatty-acyl acyl carrier protein (ACP) ligases. After activation of the fatty acyl and its ligation to the first ACP encoded by puwD, it is forwarded directly to the PKS machinery. The first unimodular PKS (encoded by puwB) contains a canonical sequence of acyltransferase, dehydratase, methyltransferase, enoylreductase and ketoreductase domains, resulting in an α-methylated product. The next enzyme (PuwE) is an unusual hybrid comprising one PKS module, a putative aminotransferase and a monooxygenase domain, and a terminal single NRPS module. The PKS module solely elongates the acyl chain with no additional modifications; however, the aminotransferase and monooxygenase located between the PKS and NRPS modules of the enzyme apparently catalyze the tailoring of the chain into a 3-amino-2-hydroxy-4-methyl-acyl typical for this group of lipopeptides. The predicted aminotransferase and monooxygenase domains show sequence similarity (50–70%) to several class-III aminotransferases and flavin-utilizing monooxygenases from cyanobacteria. The terminal NRPS module includes an A-domain activating valine as the first amino acid member of the oligopeptide ring. Valine was chosen as the most probable amino acid based on a nearest-neighbor search in NRPSPredictor2 (90%); the closest blast hits were uncharacterized cyanobacterial A-domains. Subsequently, the intermediate is transferred to another NRPS enzyme (PuwF) that comprises two modules, each adding one amino acid - dehydrothreonine and asparagine/glutamine. The A-domain putatively responsible for the incorporation of dehydrothreonine shows an amino acid-specific structural motif with considerable similarity to known threonine-activating A-domains (100% threonine in nearest-neighbor search, 64–66% pairwise identity to McnB and OciE, which are involved in cyanopeptolin synthesis in Microcystis and Planktothrix). Because no additional tailoring enzymes with amino-acid dehydration function were found in the pathway, this domain is possibly specific directly for dehydrothreonine. The second A-domain of PuwF was recovered as specific (100%) to asparagine in the nearest-neighbor analysis; however, the closest, weakly similar blast hit (54% amino acid sequence identity) was the glutamine-activating McnA protein included in cyanopeptolin biosynthetic pathways. Considering the puwainaphycin F/G variants reported in this study, the A-domain appears to accept both asparagine and glutamine as a substrate at a defined rate, as further demonstrated by MS measurements. The following biosynthesis steps are catalyzed by the puwG product that comprises three NRPS modules predicted to incorporate dehydrothreonine, asparagine and alanine. The dehydrothreonine-incorporating module is highly identical (97%) to that in PuwF, as described earlier. The other two modules activate asparagine and alanine, based on a nearest neighbor search (100%). The first shows similarity (55%) to the asparagine-activating A-domain in NosC nostopeptolide synthetase from Nostoc, and the second is similar (63% identity) to the alanine-activating domain in the jamaicamide synthesis gene JamO (Lyngbya majuscula). The last module of PuwG includes an epimerization domain consistent with the occurrence of both optical isomers (L/D-alanine) in the puwainaphycin molecules. The addition of threonine, the next member of the oligopeptide ring, is catalyzed by another protein (PuwH) that possesses a single NRPS module (100% threonine in a nearest-neighbor search, 65% identity with threonine-incorporating OciE). The last enzyme involved in the peptidyl elongation is PuwA. It clearly comprises two NRPS modules, the first containing a specific motif in the A-domain predicted to activate asparagine (90% asparagine in a nearest-neighbor search, 57% identity with NosC asparagine-activating A-domain) and a methyltransferase domain obviously linked to N-methylation of that asparagine. The second module is predicted to incorporate proline (80% proline in a nearest-neighbor search, 63% identity with NpnC proline-activating domain involved in nostophycin synthesis) and a thioesterase domain in its terminal part that cleaves the finished puwainaphycin chain from the peptidyl carrier protein, thus promoting its cyclization.


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)

Proposed biosynthesis of puwainaphycins.A, adenylation domain; ACP, acyl carrier protein; AmT, aminotransferase; AT, acyltransferase; C, condensation domain; DH, dehydratase; E, epimerase; ER, enoylreductase; FAAL, fatty acyl-AMP ligase; MT, methyltransferase; NRPS, non-ribosomal peptide synthetase; KR, ketoreductase; KS, ketosynthetase; Ox, monooxygenase; PCP, peptidyl carrier protein; PKS, polyketide synthetase; TE, thioesterase.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111904-g002: Proposed biosynthesis of puwainaphycins.A, adenylation domain; ACP, acyl carrier protein; AmT, aminotransferase; AT, acyltransferase; C, condensation domain; DH, dehydratase; E, epimerase; ER, enoylreductase; FAAL, fatty acyl-AMP ligase; MT, methyltransferase; NRPS, non-ribosomal peptide synthetase; KR, ketoreductase; KS, ketosynthetase; Ox, monooxygenase; PCP, peptidyl carrier protein; PKS, polyketide synthetase; TE, thioesterase.
Mentions: The reconstructed course of puwainaphycin biosynthesis, as visualized in Figure 2, starts with the puwC gene product. This protein contains conserved catalytic domains typical for fatty acyl-AMP ligases (FAALs), enzymes that activate a fatty acyl by adenylation to subsequently serve as a substrate for polyketide synthetases. PuwC also showed a relatively high amino acid sequence identity (approximately 60%) to several annotated cyanobacterial fatty-acyl acyl carrier protein (ACP) ligases. After activation of the fatty acyl and its ligation to the first ACP encoded by puwD, it is forwarded directly to the PKS machinery. The first unimodular PKS (encoded by puwB) contains a canonical sequence of acyltransferase, dehydratase, methyltransferase, enoylreductase and ketoreductase domains, resulting in an α-methylated product. The next enzyme (PuwE) is an unusual hybrid comprising one PKS module, a putative aminotransferase and a monooxygenase domain, and a terminal single NRPS module. The PKS module solely elongates the acyl chain with no additional modifications; however, the aminotransferase and monooxygenase located between the PKS and NRPS modules of the enzyme apparently catalyze the tailoring of the chain into a 3-amino-2-hydroxy-4-methyl-acyl typical for this group of lipopeptides. The predicted aminotransferase and monooxygenase domains show sequence similarity (50–70%) to several class-III aminotransferases and flavin-utilizing monooxygenases from cyanobacteria. The terminal NRPS module includes an A-domain activating valine as the first amino acid member of the oligopeptide ring. Valine was chosen as the most probable amino acid based on a nearest-neighbor search in NRPSPredictor2 (90%); the closest blast hits were uncharacterized cyanobacterial A-domains. Subsequently, the intermediate is transferred to another NRPS enzyme (PuwF) that comprises two modules, each adding one amino acid - dehydrothreonine and asparagine/glutamine. The A-domain putatively responsible for the incorporation of dehydrothreonine shows an amino acid-specific structural motif with considerable similarity to known threonine-activating A-domains (100% threonine in nearest-neighbor search, 64–66% pairwise identity to McnB and OciE, which are involved in cyanopeptolin synthesis in Microcystis and Planktothrix). Because no additional tailoring enzymes with amino-acid dehydration function were found in the pathway, this domain is possibly specific directly for dehydrothreonine. The second A-domain of PuwF was recovered as specific (100%) to asparagine in the nearest-neighbor analysis; however, the closest, weakly similar blast hit (54% amino acid sequence identity) was the glutamine-activating McnA protein included in cyanopeptolin biosynthetic pathways. Considering the puwainaphycin F/G variants reported in this study, the A-domain appears to accept both asparagine and glutamine as a substrate at a defined rate, as further demonstrated by MS measurements. The following biosynthesis steps are catalyzed by the puwG product that comprises three NRPS modules predicted to incorporate dehydrothreonine, asparagine and alanine. The dehydrothreonine-incorporating module is highly identical (97%) to that in PuwF, as described earlier. The other two modules activate asparagine and alanine, based on a nearest neighbor search (100%). The first shows similarity (55%) to the asparagine-activating A-domain in NosC nostopeptolide synthetase from Nostoc, and the second is similar (63% identity) to the alanine-activating domain in the jamaicamide synthesis gene JamO (Lyngbya majuscula). The last module of PuwG includes an epimerization domain consistent with the occurrence of both optical isomers (L/D-alanine) in the puwainaphycin molecules. The addition of threonine, the next member of the oligopeptide ring, is catalyzed by another protein (PuwH) that possesses a single NRPS module (100% threonine in a nearest-neighbor search, 65% identity with threonine-incorporating OciE). The last enzyme involved in the peptidyl elongation is PuwA. It clearly comprises two NRPS modules, the first containing a specific motif in the A-domain predicted to activate asparagine (90% asparagine in a nearest-neighbor search, 57% identity with NosC asparagine-activating A-domain) and a methyltransferase domain obviously linked to N-methylation of that asparagine. The second module is predicted to incorporate proline (80% proline in a nearest-neighbor search, 63% identity with NpnC proline-activating domain involved in nostophycin synthesis) and a thioesterase domain in its terminal part that cleaves the finished puwainaphycin chain from the peptidyl carrier protein, thus promoting its cyclization.

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