Recognition of Ferric Catecholates by FepA

Annamalai, Rajasekaran; Jin, Bo; Cao, Zhenghua; Newton, S. A.; Klebba, Phillip E.

doi:10.1128/jb.186.11.3578-3589.2004

Cited by 65 publications

(91 citation statements)

References 68 publications

(90 reference statements)

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“…His-420, predicted to be in the extracellular loop L7, is clearly required for initial heme coordination and stabilization. It has previously been shown that the L7 loop is critical for siderophore binding in FepA of E. coli (50). Similarly, His-86, which is predicted to be on the extracellular face of the N-terminal plug, is consistent with a higher affinity binding site located within the barrel.…”

Section: Discussionsupporting

confidence: 56%

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

Burkhard

Wilks

2007

Journal of Biological Chemistry

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Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.The ability to acquire iron is essential for the survival and virulence of the large majority of pathogenic bacteria (1). Bacterial pathogens have therefore evolved sophisticated systems to acquire a variety of iron complexes (2). Most bacteria excrete siderophores, high affinity low molecular weight chelators that sequester iron for internalization via receptor-mediated uptake. In addition, many pathogens obtain iron by utilizing the host's iron and heme 2 -containing proteins (2, 3). These complex transport systems in Gram-negative bacteria all require a TonB-dependent high affinity outer membrane receptor to facilitate transport across the outer membrane (4). The transport of heme across the outer membrane is driven by coupling the cytoplasmic membrane potential via the TonB-ExbBD complex to the receptor. Following transport across the outer membrane, a periplasmic-binding protein shuttles the heme to the cytoplasmic ATPase/permease, where the heme is internalized and further utilized (5-7).A number of Gram-negative pathogens have been shown to utilize a wide spectrum of iron and heme-containing proteins, including Yersinia sp. (8 -10), Neisseria sp. (11-13), Vibrio sp. (14), and the opportunistic pathogen Pseudomonas aeruginosa (15). The well characterized Yersinia entercolitica heme operon (hemRSTUV) encodes the outer membrane receptor HemR and the periplasmic...

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Section: Discussionsupporting

confidence: 56%

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

Burkhard

Wilks

2007

Journal of Biological Chemistry

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“…Although there is no structure for the siderophore complex of FepA from E. coli, several binding and mutation studies have been reported (46)(47)(48). These studies suggest a biphasic binding kinetic with an initial binding site in the loop extremities and a secondary site deeper inside the barrel leading to the translocation.…”

Section: Discussionmentioning

confidence: 99%

“…The presumed second binding site, on top of the plug domain, is highly conserved between the two PirAs and FepA. Deletion of loop NL1 or NL3 in FepA or mutation of the conserved Trp101 in NL3 (corresponding to Trp111 and Trp110 in Ab-PirA and Pa-PirA) drastically decreased the uptake of ferric enterobactin (48,49). Other key residues known to be important in FepA are Tyr260 of L3 (262 in Ab-PirA and 264 in Pa-PirA), Arg316 on ␤7 (Ab-PirA R320 and Pa-PirA R321), and Tyr478 of L7 (Ab-PirA Tyr478 and Pa-PirA Tyr479), which are conserved in PirA.…”

Section: Discussionmentioning

confidence: 99%

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

Moynié

Lüscher

Rolo

et al. 2017

Antimicrob Agents Chemother

103

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The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we investigated the mechanism of action of these molecules in A. baumannii. We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4-to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4-to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gramnegative pathogens.

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“…Under the same conditions, the absence of quenching with Fe(III)-S4 and (Fig. 2B), which is Ͼ10 3 -fold weaker than the affinity of the IroN homolog FepA for its siderophore ligand, Ent (21). In a separate qualitative cocrystallization binding assay, millimolar Lcn2 was mixed with excess ferric siderophore, and the degree of ligand binding was determined by the color of the crystals as judged by visual inspection.…”

Section: Resultsmentioning

confidence: 99%

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

Fischbach

Lin

Zhou

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

274

238

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Numerous bacteria cope with the scarcity of iron in their microenvironment by synthesizing small iron-scavenging molecules known as siderophores. Mammals have evolved countermeasures to block siderophore-mediated iron acquisition as part of their innate immune response. Secreted lipocalin 2 (Lcn2) sequesters the Escherichia coli siderophore enterobactin (Ent), preventing E. coli from acquiring iron and protecting mammals from infection by E. coli. Here, we show that the iroA gene cluster, found in many pathogenic strains of Gram-negative enteric bacteria, including E. coli, Salmonella spp., and Klebsiella pneumoniae, allows bacteria to evade sequestration of Ent by Lcn2. We demonstrate that Cglucosylated derivatives of Ent produced by iroA-encoded enzymes do not bind purified Lcn2, and an iroA-harboring strain of E. coli is insensitive to the growth inhibitory effects of Lcn2 in vitro. Furthermore, we show that mice rapidly succumb to infection by an iroA-harboring strain of E. coli but not its wild-type counterpart, and that this increased virulence depends on evasion of host Lcn2. Our findings indicate that the iroA gene cluster allows bacteria to evade this component of the innate immune system, rejuvenating their Ent-mediated iron-acquisition pathway and playing an important role in their virulence.bacterial pathogens ͉ host defense ͉ innate immunity ͉ iron ͉ siderophores

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Recognition of Ferric Catecholates by FepA

Abstract: Escherichia coli

Cited by 65 publications

References 68 publications

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2

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