Tistrellabactins A and B Are Photoreactive C-Diazeniumdiolate Siderophores from the Marine-Derived Strain Tistrella mobilis KA081020-065

Abstract

The C-diazeniumdiolate group in the amino acid graminine is emerging as a new microbially produced Fe(III) coordinating ligand in siderophores, which is photoreactive. While the few siderophores reported from this class have only been isolated from soil-associated microbes, here we report the first C-diazeniumdiolate siderophores tistrellabactins A and B, isolated from the bioactive marine-derived strain Tistrella mobilis KA081020-065. The structural characterization of the tistrellabactins reveals unique biosynthetic features including an NRPS module iteratively loading glutamine residues and a promiscuous adenylation domain yielding either tistrellabactin A with an asparagine residue or tistrellabactin B with an aspartic acid residue at analogous positions. Beyond the function of scavenging Fe(III) for growth, these siderophores are photoreactive upon irradiation with UV light, releasing the equivalent of nitric oxide (NO) and an H atom from the C-diazeniumdiolate group. Fe(III)-tistrellabactin is also photoreactive, with both the C-diazeniumdiolate and the β-hydroxyaspartate residues undergoing photoreactions, resulting in a photoproduct without the ability to chelate Fe(III).

Figure 1

Select natural products with N–N bond linkages, highlighted in blue.

Figure 2

Tistrellabactins A and B are mixed-ligand siderophores isolated from the marine-derived strain T. mobilis KA081020-065. (A) BGC of tistrellabactins A and B encodes sequence homologues of grbD (mobE) and grbE (fused to the start of mobF), which are implicated in the biosynthesis of the graminine residues N-monooxygenase (MobI) and N-acetyltransferase (MobJ), which install the hydroxy and acetyl groups on l-Orn, and a TβH_Asp hydroxylase (MobD), which hydroxylates aspartic acid. (B) NRPS module breakdown of MobF, MobG, and MobH. MobG iteratively loads a second Gln residue. MobH has a promiscuous A domain, which can activate l-Asn or l-Asp, yielding tistrellabactins A and B, respectively. Abbreviations: coenzyme ligase A domain (CAL), condensation domain (C), thiolation domain (T), epimerization domain (E), thioesterase (Te).

Figure 3

(A) HPLC chromatogram trace (215 nm) showing tistrellabactin A and tistrellabactin B elute side by side in 20% aqueous MeOH + 0.05% TFA, with favored production of tistrellabactin A to tistrellabactin B. (B) Tistrellabactin A (m/z 1092.4 [M + H]⁺) and (C) tistrellabactin B (m/z 1093.4 [M + H]⁺) differ by one mass unit, and each displays an ionization mass loss of 30 Da, characteristic of C-diazeniumdiolates. (D) The structures of isolated and purified tistrellabactin A (R = NH₂) and tistrellabactin B (R = OH) were elucidated by NMR spectroscopy and MS.

Figure 4

NMR-guided structure elucidation of tistrellabactins A and B. (A) Key ¹H–¹³C HMBC and ¹H–¹H COSY correlations in tistrellabactin A. (B) Superimposed tistrellabactin A (maroon) and tistrellabactin B (blue) ¹H–¹³C HMBC correlations show three Gln NH₂ side chain ¹H pairs correlate to respective Gln Cγ. An HMBC correlation between Asn side chain NH₂¹H at 6.99 ppm and Cβ ¹³C at 34.85 ppm is not present in tistrellabactin B, which has an Asp residue in the analogous position. (C) ¹H–¹⁵N HMBC shows the presence of C-diazeniumdiolate amino acid Gra.

Figure 5

NRPS domain organization and proposed biosynthesis of tistrellabactin A. NRPS modules 4 and 5 iteratively load Gln residues via an unknown mechanism.

Figure 6

Photoreactivity of apo-tistrellabactin A. (A) The diazeniumdiolate absorbance band at 246 nm disappears with irradiation of UV light. (B) The C-diazeniumdiolate is converted to E/Z oxime isomers. (C) Proposed coordination of the apo-tistrellabactin A photoproduct to Fe(III).