Bacteria possess complex machinery for uptake of essential iron. Studying iron-uptake provides insight into requirements for bacterial growth, and for development of applications, including novel antibiotics and diagnostic tools.
The presented project investigates the binding of periplasmic binding proteins CeuE, FepB and VctP with a range of iron(III)-siderophore and siderophore-mimic compounds. CeuE was shown to bind iron(III)-n-LICAM (n= 6, 8) (N,N'-hexane-1,6-diylbis (2,3-dihydroxybenzamide and N,N'-octane-1,8-diylbis (2,3-dihydroxybenzamide)) tetradentate siderophore mimics, with Λ-configuration in both crystal and solution phase, with binding constants of 33 ±8 and 58 ± 8 nM respectively. Comparing these results to those for iron(III)-n-LICAM (n= 4, 5) (N,N'-butane-1,4-diylbis (2,3-dihydroxybenzamide) and N,N'-pentane-1,5-diylbis (2,3-dihydroxybenzamide)) revealed that the highest affinity was found for a five-atom linker. Mutagenesis of His 227 and Tyr 288 that coordinate the iron(III)-centre, proved that Tyr 288 is essential for iron(III)-n-LICAM binding to CeuE. Binding affinity is slightly reduced for all iron(III)-n-LICAM (n= 4, 5, 6, 8) ligands with mutation to His 227. CeuE-H227L-iron(III)-5-LICAM crystal structure determination revealed that iron(III)-5-LICAM bound in the Λ-configuration with one aqua-ligand.
Salmochelin mimic siderophores Sal-n-LICAM (n= 4, 5) were synthesised and iron(III)-binding established via Job plot. For both compounds, equilibria of 1:1 and 3:2 ligand:metal ratios were observed, with 3:2 predominating over time. CeuE bound both iron(III)-complexes weakly in the Λ-configuration. Iron(III)-Sal-5-LICAM bound with higher affinity (511 ±76 nM) than iron(III)-Sal-4-LICAM (15.6 ± 2.3 µM).
FepB was overexpressed, purified, and its siderophore-binding profile compared with that of CeuE and VctP. It was shown that FepB bound iron(III)-enterobactin with nanomolar affinity, and had micromolar affinity for tetradentate catecholate complexes. CeuE bound tetradentate catecholate complexes with nanomolar affinity, and iron(III)-enterobactin with micromolar affinity. VctP bound tetradentate catecholate complexes with picomolar affinity, iron(III)-enterobactin with mid-nanomolar affinity, and iron(III)-Sal-n-LICAM (n= 4, 5) with low-nanomolar affinity. All three proteins bound iron(III)-MECAM (1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) with low nanomolar affinity.
