Search

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdine PvdI, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, d-Ser- l-Arg- d-Ser- l-FoOHOrn-( l-Lys- l-FoOHOrn- l-Thr- l-Thr), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the l-Lys side chain. In aqueous solution at room temperature, the (1)H NMR spectrum of pyoverdine PvdI-Ga(III) showed clear evidence of exchange broadening. At 253 K, two distinct conformations were observed and the measurement of structural constraints was possible. The three-dimensional structures of the two PvdI-Ga(III) conformers were determined, and analysis of the structures indicates that the observed conformational exchange involves a stereoisomerization of the metal binding coordination accompanied by a change in the global shape of the siderophore. This conformational transition was further characterized by heteronuclear relaxation experiments. The possible implications of this dynamic behavior for siderophore recognition by the receptor FpvAI are discussed.

Under iron limitation, Pseudomonas aeruginosa ATCC 15692 secretes a major siderophore, pyoverdine I (PvdI). This molecule chelates iron in the extracellular medium and shuttles it into the cells via a specific outer membrane transporter, FpvAI. PvdI consists of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and containing one of the bidentate groups involved in iron chelation, linked to a peptide moiety containing the two other bidentate groups required for binding to Fe(3+). Kinetic studies, based on the fluorescence properties of this siderophore, showed that pH 8.0 was optimal for the binding of PvdI and PvdI-Fe to FpvAI. We investigated the mechanism of interaction of PvdI and PvdI-Fe with FpvAI, by synthesizing various analogues of this siderophore, determining their affinity for FpvAI in vitro and in vivo and their ability to transport iron, and interpreting the results obtained in light of the structure of FpvAI-PvdI. Our findings demonstrate that the succinyl moiety linked to the chromophore of PvdI and the first amino acid of the peptide moiety can be sterically hindered with no effect on binding or the iron uptake properties of PvdI-Fe. Moreover, the sequence and the structure of the peptide moiety of PvdI seems to be more important for the iron uptake step than for the binding of the siderophore to FpvAI. Finally, the efficiency of iron uptake and of recycling of the various PvdI analogues after iron release suggests that iron dissociates from PvdI on FpvAI or in the periplasm. All these data have serious implications for the specificity and mechanism of PvdI-mediated iron transport in P. aeruginosa.

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdin PaA, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, D-Ser-L-Arg-D-Ser-L-FoOHOrn-(L-Lys-L-FoOHOrn-L-Thr-L-Thr) (FoOHOrn: delta N-formyl-delta N-hydroxyornithine), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the L-Lys side chain. Ferric iron is chelated by the catechol group on the chromophore and the two hydroxyornithine side chains. In aqueous solution, the (1)H-NMR spectrum of pyoverdin PaA-Ga(III), in which Ga(III) is used instead of Fe(III) for spectroscopic purposes, showed clear evidence of exchange broadening, preventing further structural characterization. The use of cryo-solvents allowed measurements to be made at temperatures as low as 253 K where two distinct conformations with roughly equivalent populations could be observed. (13)C and (15)N labeling of pyoverdin PaA enabled complete assignment of both forms of pyoverdin PaA-Ga(III) at 253 and 267 K, using triple-resonance multidimensional NMR experiments commonly applied to doubly labeled proteins., ((c) 2005 Wiley Periodicals, Inc. Biopolymers 79: 139-149, 2005.)

In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins.

Among the fluorescent Pseudomonas species, Pseudomonas putida is a rare case of a nitrogen-fixing bacterium that transforms nitrogen into ammonia. When grown under iron-deficient conditions, it produces two major pyoverdins: pyoverdin G4R and pyoverdin G4RA. Their primary structures have been established using FAB-MS and one- and two-dimensional 15N, 13C, and 1H NMR on both the unlabeled and 15N-labeled compounds [Salah El Din, A. L. M., et al. (1997) Tetrahedron 53, 12539-12552]. The two pyoverdins have a common chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline. The chromophore is bound to the linear heptapeptide L-Asp-L-Orn-D-beta-threo-OHAsp-L-Dab-Gly-L-Ser-L-cyclo-OHOrn . Circular dichroism spectra suggest that the absolute configuration of the metal complex is Delta. The three-dimensional structure in solution of pyoverdin G4R-Ga(III) was determined after interpretation of two-dimensional 1H NMR spectra recorded at 283 and 303 K. The complex is tightly defined with a compact structure with a Delta absolute configuration. The site of complexation of the metal ion is found to be located on the surface of the molecule, showing that the ion can be released without large conformational changes, while the polar groups of the peptide chain, which may be responsible for the recognition of the receptor, are placed on the opposite side of the overall shape. The three-dimensional structure of pyoverdin G4R-Ga(III) is compared with those of other pyoverdins, and the role of the structure in iron uptake is discussed.

Echistatin is a 49 amino acid protein isolated from the venom of a viper (Echis carinatus) and is one of the smallest natural adhesive ligands that interacts with integrin-type receptors through an Arg-Gly-Asp (RGD) sequence. The structure of echistatin in aqueous solution has been determined by nuclear magnetic resonance spectroscopy. Nuclear Overhauser spectra yielded 490 interatomic distance constraints, which were used in distance geometry calculations. The chain is shown to fold in a series of irregular loops to form a rigid core stabilized by four cystine cross-links. From this core an irregular hairpin and the C-terminus protrude. The core and the hairpin are further stabilized by a network of hydrogen bonds. The RGD sequence is located in a mobile loop at the tip of the hairpin. The mobility and its significance for activity are discussed.