Reduction-cleavable desferrioxamine B pulldown system enriches Ni(ii)-superoxide dismutase from a Streptomyces proteome

Two resins with the hydroxamic acid siderophore desferrioxamine B (DFOB) immobilised as a free ligand or its Fe(iii) complex were prepared to screen the Streptomyces pilosus proteome for proteins involved in siderophore-mediated Fe(iii) uptake. The resin design included a disulfide bond to enable the release of bound proteins under mild reducing conditions. Proteomics analysis of the bound fractions did not identify proteins associated with siderophore-mediated Fe(iii) uptake, but identified nickel superoxide dismutase (NiSOD), which was enriched on the apo-DFOB-resin but not the Fe(iii)-DFOB-resin or the control resin. While DFOB is unable to sequester Fe(iii) from sites deeply buried in metalloproteins, the coordinatively unsaturated Ni(ii) ion in NiSOD is present in a surface-exposed loop region at the N-terminus, which might enable partial chelation. The results were consistent with the notion that the apo-DFOB-resin formed a ternary complex with NiSOD, which was not possible for either the coordinatively saturated Fe(iii)-DFOB-resin or the non-coordinating control resin systems. In support, ESI-TOF-MS measurements from a solution of a model Ni(ii)-SOD peptide and DFOB showed signals that correlated with a ternary Ni(ii)-SOD peptide–DFOB complex. Although any biological implications of a DFOB–NiSOD complex are unclear, the work shows that the metal coordination properties of siderophores might influence an array of metal-dependent biological processes beyond those established in iron uptake.


HRMS (compound characterisation)
. HRMS analysis was conducted on a Thermofisher Vanquish Horizon UHPLC coupled to a Thermofisher Q-Exactive HFX Hybrid Quadrupole-Orbitrap mass spectrometer.Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile.An Agilent Zorbax Eclipse XDB-C18 column (150 mm x 2.1 mm I.D., 3.5 µm) maintained at 30 °C with a constant flow rate of 0.2 mL/min was employed.A sample injection volume of 5 µL was used and the solvent program was as follows: a linear gradient of 5-95% solvent B over 25 min followed by 95% solvent B held for 5 min.Solvent B was then held at 5% for an additional 5 min to re-equilibrate for the following injection.The mass spectrometer was operated in positive ion-mode using electrospray ionisation (ESI) with a mass range of 100-1500 m/z.A spray voltage of 3.5 kV was used and the capillary temperature maintained at 300 °C.

HPLC (compound purification).
Preparative HPLC purification was undertaken on a Shimadzu LC-20 series high performance liquid chromatography system with two LC-20AP preparative pumps, a SIL-10AP autosampler and a SPD-20A photo-diode array detector.A Shim-pack GIS-C18 column (150 x 20 ID, 5 µm) was used at 20 mL/min.Mobile phase A was 0.1 % formic acid in Milli-Q water and mobile phase B was 0.1 % formic acid in acetonitrile with solvent programs as stated below for individual compounds.

Preparative scale synthesis of N-DFOB-3-mercaptopropanamide (DFOB-SH) (2).
Triethylamine (67 µL, 0.48 mmol.) and DTSP (53 mg, 0.13 mmol.) were added to a solution of DFOB mesylate salt (151 mg, 23 mmol.) in anhydrous DMF (2 mL) under nitrogen.The resulting mixture was heated at 70 °C for 16 h under nitrogen.The solvent was removed in vacuo and the crude material reconstituted in water.The suspension was pH adjusted to 10 with 1 M sodium hydroxide solution.TCEP hydrochloride (0.5 mL, 0.5 M aqueous solution, 0.25 mmol.) was added to the aqueous mixture and left to stir at room temperature under nitrogen for 4 h.The solvent was removed in vacuo and the crude material reconstituted in 1 M sodium hydroxide solution for purification by preparative HPLC (5-60 % mobile phase B over 20 min) yielding the desired product (2) as a white powder (50 mg, 34%).

Semi-preparative scale synthesis of N-methyl-3-mercaptopropanamide (MA-SH) (3).
Methylamine hydrochloride (35 mg, 0.52 mmol) and DTSP (105 mg, 0.26 mmol) was dissolved in a miscible solution of 5.5% water in acetone.Triethylamine (72 µL, 0.25 mmol) was added and the reaction stirred at room temperature for 24 h.The solution was then concentrated in vacuo and redissolved in water.2-Mercaptoethanol (36 µL, 0.52 mmol) was then added into the crude mixture and the reaction stirred under nitrogen overnight.The resulting solution was then HPLC purified (0% mobile phase B held for 3 min, 0-20% mobile phase B over 12 min) and the collected fractions dried in vacuo.Qualitative LC-MS analysis confirmed the isolation of the desired free thiol however, NMR analysis showed MA-SH (3) dimerised on standing to regenerate (MA)2-SS (3a).The isolated material was spectroscopically characterised as a mixture of (MA)2-SS (3a) and MA-SH (3).

Fig. S13 .
Fig. S13.LC-MS trace from reaction mixtures containing 3a reported as (a) TIC, with experimental (black) MS signals from the peak at (b) 6.5 min, with calculated signals ([M+H] + adduct) in grey.LC-MS trace from reaction mixtures of 3a and TCEP containing 3 reported as (c) TIC, with experimental (black) MS signals from the peak at (d) 2.4 min, with calculated signals ([M+H] + adduct) in grey.

Fig. S16 .
Fig. S16.Absorbance (λ = 470 nm) from culture medium inoculated with S. pilosus sub-sampled at 2-d intervals over 0-10 d, with aliquots mixed with Fe(III) prior to analysis.Error bars represent the range for two replicate measurements.