Highly diverse cyanobactins in strains of the genus Anabaena

Abstract

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.

FIG. 1.

Anacyclamide biosynthetic pathway in Anabaena sp. 90. (A) acy gene cluster from Anabaena sp. 90. The genes indicated by black and gray arrows have homology to genes present in other cyanobactin gene clusters. The acyE gene encoding the precursor peptide is indicated by a gray arrow and is enclosed in a box. The genes indicated by open arrows encode conserved and hypothetical proteins without known or predicted functions. (B) AcyE peptide precursor from Anabaena sp. 90 with the hypervariable region of the 49-amino-acid protein encoding the mature anacyclamide shaded, indicating the position of cleavage and macrocyclization. The eight conserved amino acids in the AcyE leader peptide and other known cyanobactin precursor leader peptides are indicated arrowheads. (C) Structure of the decapeptide anacyclamide A10 from Anabaena sp. 90.

FIG. 2.

Peptide precursor proteins from Nostoc spongiaeforme var. tenue (TenE), Lyngbya aesturarii PCC 8106 (LynE) (4), Prochloron (PatE) (20), Microcystis aeruginosa PCC7806 (McaE1), Microcystis aeruginosa NIES 298 (McaE2) (28), Prochloron (TruE) (4), Trichodesmium erythraeum IMS 101 (TriG) (24), and Anabaena sp. 90 (AcyE). The homologous amino acids are indicated by asterisks. The areas of the precursor protein which form the mature cyanobactin are shaded.

FIG. 3.

Shift in mass detected from comparison of mass spectra for extracts of Anabaena sp. strain SYKE 844B cultivated on normal medium (³²S) (spectrum below the line) and on medium containing ³⁴S (spectrum above the line), presented as mirror images. The m/z 764 peak is the protonated ion from ³⁴S-labeled anacyclamide B7, and the m/z 786 peak is its Na adduct; the corresponding ³²S ions are the m/z 762 and 784 ions, respectively.

FIG. 4.

Ion chromatograms for the protonated biosynthetic and synthetic reference anacyclamides A10, C10, A15, and B7. Ion masses and the anacyclamides are shown in Table 1. Chromatogram 1, Anabaena strain 90; chromatogram 2, Anabaena strain 0TU33S16; chromatogram 3, Anabaena strain SYKE971/6; chromatogram 4, Anabaena strain 299A; chromatogram 5, Anabaena strain SYKE748A; chromatogram 6, Anabaena strain 37; chromatogram 7, Anabaena strain SYKE 844B. The peak eluting later in the ion chromatogram of strain 0TU33S16 originated from a cyclic peptide belonging to another peptide family.

FIG. 5.

Comparison of the product ion spectra for the m/z 762.4 (MH⁺) and m/z 760.4 (M-H⁻) molecular ions of synthetic and biosynthetic (Anabaena SYKE 844B) anacyclamide B7.

FIG. 6.

Comparison of biosynthetic anacyclamide A10 of Anabaena sp. 90 to a synthetic standard and anacyclamide A10 produced in E. coli. (A to C) Mass spectra of (A) protonated biosynthetic anacyclamide produced by Anabaena sp. 90, (B) synthetic anacyclamide, and (C) biosynthetic anacyclamide produced by E. coli(pNLA90). (D and E) Mass spectra of (D) sodiated anacyclamide A10 from Anabaena sp. 90 and (E) a synthetic standard.

FIG. 7.

Chromatographic and mass spectrometric data showing the presence of a structural unit with a mass of 68 mass units in anacyclamide A9P. (A) Ion chromatograms of m/z 1114.6 and m/z 1046.5 ions. (B) Product ion spectrum for protonated derivatized anacyclamide A9P m/z 1114.6 ion. (C and D) Product ion spectra for the m/z 1046.5 protonated ion formed (C) in the ion source and (D) in the ion trap.