title: Pseudoaeruginosins, nonribosomal peptides in Nodularia spumigena.
abstract: Nodularia spumigena is a filamentous cyanobacterium that forms toxic blooms in brackish waters around the world through the production of the pentapeptide toxin nodularin. This cyanobacterium also produces large amounts of protease inhibitors belonging to the aeruginosin and spumigin families. Here we report the discovery of previously unknown protease inhibitors, pseudoaeruginosins NS1 (1) and NS2 (2), from 33 strains of N. spumigena isolated from the Baltic Sea. Pseudoaeruginosin NS1 (1) and NS2 (2) contain hexanoic acid, tyrosine, 4-methylproline, and argininal/argininol. The chemical structure of the two pseudoaeruginosins was verified by thorough comparison of the liquid chromatography-mass spectrometry (LC-MS) analyses of the extracts from the N. spumigena strains with synthetic peptides. The structures of the synthetic pseudoaeruginosins were confirmed using nuclear magnetic resonance spectroscopy. Surprisingly, the structure of pseudoaeruginosin NS1 (1) and NS2 (2) combines features of both aeruginosins and spumigins, suggesting that they have been produced through the joint action of both the spumigin and aeruginosin biosynthesis pathways. We screened with polymerase chain reaction and LC-MS 68 N. spumigena strains from the Baltic Sea and Australia. Pseudoaeruginosins were present in half of the Baltic Sea strains but were not found from the Australian strains. The production of pseudoaeruginosin seems to be coupled to the production of aeruginosins and 4-methylproline-containing spumigins. Pseudoaeruginosin NS1 was found to be as potent trypsin inhibitor as the most potent aeruginosins and spumigins with an IC50 of 0.19 ± 0.04 μM. This finding suggests that cooperation between the spumigin and aeruginosin biosynthetic pathways results in hybrid pseudoaeruginosin peptides.
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: Liwei
authors.initials: L
authors.lastname: Liu
authors.affiliation: ‡Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégaten 41, 5007 Bergen, Norway.
authors.forename: Adnan
authors.initials: A
authors.lastname: Budnjo
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: Jouni
authors.initials: J
authors.lastname: Jokela
authors.affiliation: ‡Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégaten 41, 5007 Bergen, Norway.
authors.forename: Bengt Erik
authors.initials: BE
authors.lastname: Haug
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: David P
authors.initials: DP
authors.lastname: Fewer
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: Matti
authors.initials: M
authors.lastname: Wahlsten
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: Leo
authors.initials: L
authors.lastname: Rouhiainen
authors.affiliation: §Program in Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, P.O. Box 65, FI-00014 Helsinki, Finland.
authors.forename: Perttu
authors.initials: P
authors.lastname: Permi
authors.affiliation: ‡Department of Chemistry and Centre for Pharmacy, University of Bergen, Allégaten 41, 5007 Bergen, Norway.
authors.forename: Torgils
authors.initials: T
authors.lastname: Fossen
authors.affiliation: †Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, P.O. Box 56, Viikki Biocenter, Viikinkaari 9, FI-00014 Helsinki, Finland.
authors.forename: Kaarina
authors.initials: K
authors.lastname: Sivonen
mesh_terms: D001315:Australia; D001426:Bacterial Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D001454:Baltic States; D002853:Chromatography, Liquid; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry; D007700:Kinetics; D046932:Nodularia / Q000737:chemistry* / Q000378:metabolism; D009792:Oceans and Seas; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000302:isolation & purification; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D010456:Peptides, Cyclic; D011480:Protease Inhibitors; C000603226:pseudoaeruginosin NS1; C000603227:pseudoaeruginosin NS2; C063998:nodularin; D002918:Chymotrypsin; D014357:Trypsin