Structural and Stereochemical Elucidation of Cyanochelin C, a Siderophore Associated with Novel Class of Cyanobacterial Acyl Hydrolases

Key results

1. ShownCyanochelin C is a seven-residue siderophore, Val-methyloxazoline-Lys-β-Ala-β-OH-Asp-Gly-DHABA (β-hydroxyaspartate; 2,4-dihydroxy-3-aminobutyric acid), with nine stereocenters and no acyl chain. It forms an iron complex and was recovered from the culture medium.

   High-resolution MS (HRMS) gave [M+H]+ m/z 705.3429 (C28H49N8O13) and an iron complex at m/z 758.2536. The MS/MS series (m/z 634.2649, 523.2367, 395.1406, 324.1036, 193.0817) followed the predicted adenylation-domain order. 700 MHz 1D/2D NMR (COSY, HSQC, HMBC) placed the methyloxazoline and the C-terminal DHABA. Yield was 17.6 mg from 3 L of spent medium. · Identification and isolation of Cyanochelin C; Structure elucidation of cyanochelin C; Tables

2. ShownAdvanced Marfey's analysis established L-Val, L-Thr (2S,3R) and D-Lys. This agrees with the gene cluster, whose lysine module carries an epimerization domain.

   L-FDAA derivatives of the acid hydrolysate (10 μg of compound) were compared by HPLC-HRMS retention time with L-DAA and D-DAA reference derivatives prepared from authentic L-Lys, L-Val and L-Thr. D-Lys matches the epimerization step predicted for the ccsF lysine module. · Structure elucidation of cyanochelin C; Advanced Marfey’s analysis; Biosynthetic gene cluster encoding production of…

3. SuggestedJ-based configuration analysis, together with cluster evidence, assigned threo (2R,3S)-DHABA and L-erythro (8S,9R) β-OH-Asp. According to the literature the paper cites, L-erythro β-OH-Asp had previously been confirmed only in imaqobactin.

   DHABA: 3J(H2,H3) of 2 Hz, with small 3J(C,H) and 2J(C,H) couplings from HMBC slices and HSQC-HECADE. β-OH-Asp: 3J(H8,H9) of 5.2 Hz, with a small H-8/C-10 and a medium H-9/C-7 3J(C,H); this pattern fits only Murata's erythro model, although the H-9/C-8 2J(C,H) (−4.1 Hz) was smaller than that model expects (H-8/C-9 was −4.2 Hz). The β-OH-Asp module lacks an epimerization domain, implying the L form. · Structure elucidation of cyanochelin C; Discussion

4. SuggestedThe cluster has a FAAL starter, yet the isolated siderophore carries no acyl chain. The oppositely oriented cluster gene ccsQ encodes a PvdQ-homologous acylase proposed to remove the chain, and an AlphaFold3 model places a fatty acid with its carboxyl toward the active site.

   antiSMASH found the cluster in M. chilensis ATA2-1-KO14 and M. californica WJT36-NPBG1 (89,6% nucleotide similarity); it sits beside a siderophore transporter cassette. In a tree of characterised N-terminal nucleophile hydrolases, CcsQ is closest to cephalosporin/glutaryl-7-ACA acylases, then PvdQ and the marinobactin acylases. Model scores: pTM 0.96; myristic acid ipTM 0.96; peptide ipTM 0.69. · Biosynthetic gene cluster encoding production of…; Characterization of siderophore maturation…; Phylogenetic analyses and Sequence-Similarity…; Protein modelling

5. ShownIn a sequence-similarity network of 997 BLAST hits, cyanobacterial CcsQ homologues group apart from other phyla. Within cyanobacteria, a well-supported acylase lineage is tied to siderophore clusters and is separate from one that mostly lacks them; the authors read its branching as largely vertical inheritance.

   An acylase is present in 22 out of 31 earlier-published cyanochelin-like clusters. A PSI-BLAST seeded with these 22 found 259 cyanobacterial sequences in the first 1 000 hits. Tree: 292 taxa and 642 aligned positions (Acidobacteriota outgroup). ∼47% of 'cyanobacteriota 1' acylases neighbour cyanochelin-like clusters; six (∼8%) in 'cyanobacteriota 2' neighbour NRPS clusters without siderophore signs. · Distribution of N-terminal nucleophile acyl…; Phylogenetic analyses and Sequence-Similarity…; Phylogenetic analysis of CcsQ-like N-terminal…