The Experts below are selected from a list of 2007 Experts worldwide ranked by ideXlab platform
Elizabeth M Nolan - One of the best experts on this subject based on the ideXlab platform.
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design solid phase synthesis and evaluation of Enterobactin analogs for iron delivery into the human pathogen campylobacter jejuni
2018Co-Authors: Cristina Y Zamora, Amael G E Madec, Wilma Neumann, Elizabeth M Nolan, Barbara ImperialiAbstract:Abstract The human enteropathogen Campylobacter jejuni , like many bacteria, employs siderophores such as Enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as inspiration for the development of synthetic carriers that may effectively transport cargo into Gram-negative bacteria. In this work, we have developed a facile and robust microscale assay to measure growth recovery of C. jejuni NCTC 11168 in liquid culture as a result of ferric iron uptake. In parallel, we have established the solid-phase synthesis of catecholamide compounds modeled on Enterobactin fragments. Applying these methodological developments, we show that small synthetic iron chelators of minimal dimensions provide ferric iron to C. jejuni with equal or greater efficiency than Enterobactin.
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determination of the molecular structures of ferric Enterobactin and ferric enantioEnterobactin using racemic crystallography
2017Co-Authors: Timothy C Johnstone, Elizabeth M NolanAbstract:Enterobactin is a secondary metabolite produced by Enterobacteriaceae for acquiring iron, an essential metal nutrient. The biosynthesis and utilization of Enterobactin permits many Gram-negative bacteria to thrive in environments where low soluble iron concentrations would otherwise preclude survival. Despite extensive work carried out on this celebrated molecule since its discovery over 40 years ago, the ferric Enterobactin complex has eluded crystallographic structural characterization. We report the successful growth of single crystals containing ferric Enterobactin using racemic crystallization, a method that involves cocrystallization of a chiral molecule with its mirror image. The structures of ferric Enterobactin and ferric enantioEnterobactin obtained in this work provide a definitive assignment of the stereochemistry at the metal center and reveal secondary coordination sphere interactions. The structures were employed in computational investigations of the interactions of these complexes with tw...
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Enterobactin mediated delivery of β lactam antibiotics enhances antibacterial activity against pathogenic escherichia coli
2014Co-Authors: Tengfei Zheng, Elizabeth M NolanAbstract:The design, synthesis, and characterization of Enterobactin-antibiotic conjugates, hereafter Ent-Amp/Amx, where the β-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized Enterobactin scaffold via a stable poly(ethylene glycol) linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide enhanced antibacterial activity against Escherichia coli strains, including uropathogenic E. coli CFT073 and UTI89, enterohemorrhagic E. coli O157:H7, and enterotoxigenic E. coli O78:H11, compared to the parent β-lactams. Studies with E. coli K-12 derivatives defective in ferric Enterobactin transport reveal that the enhanced antibacterial activity observed for this strain requires the outer membrane ferric Enterobactin transporter FepA. A remarkable 1000-fold decrease in minimum inhibitory concentration (MIC) value is observed for uropathogenic E. coli CFT073 relative to Amp/Amx, and time-kill kinetic studies demonstrate that Ent-Amp/Amx kill this strain more rapidly at 10-fold lower concentrations than the parent antibiotics. Moreover, Ent-Amp and Ent-Amx selectively kill E. coli CFT073 co-cultured with other bacterial species such as Staphylococcus aureus, and Ent-Amp exhibits low cytotoxicity against human T84 intestinal cells in both the apo and iron-bound forms. These studies demonstrate that the native Enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens utilizing Enterobactin for iron acquisition.
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siderophore mediated cargo delivery to the cytoplasm of escherichia coli and pseudomonas aeruginosa syntheses of monofunctionalized Enterobactin scaffolds and evaluation of Enterobactin cargo conjugate uptake
2012Co-Authors: Tengfei Zheng, Justin L Bullock, Elizabeth M NolanAbstract:The design and syntheses of monofunctionalized Enterobactin (Ent, l- and d-isomers) scaffolds where one catecholate moiety of Enterobactin houses an alkene, aldehyde, or carboxylic acid at the C5 position are described. These molecules are key precursors to a family of 10 Enterobactin–cargo conjugates presented in this work, which were designed to probe the extent to which the Gram-negative ferric Enterobactin uptake and processing machinery recognizes, transports, and utilizes derivatized Enterobactin scaffolds. A series of growth recovery assays employing Enterobactin-deficient E. coli ATCC 33475 (ent-) revealed that six conjugates based on l-Ent having relatively small cargos promoted E. coli growth under iron-limiting conditions whereas negligible-to-no growth recovery was observed for four conjugates with relatively large cargos. No growth recovery was observed for the Enterobactin receptor-deficient strain of E. coli H1187 (fepA-) or the Enterobactin esterase-deficient derivative of E. coli K-12 JW0...
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siderophore mediated cargo delivery to the cytoplasm of escherichia coli and pseudomonas aeruginosa syntheses of monofunctionalized Enterobactin scaffolds and evaluation of Enterobactin cargo conjugate uptake
2012Co-Authors: Tengfei Zheng, Justin L Bullock, Elizabeth M NolanAbstract:The design and syntheses of monofunctionalized Enterobactin (Ent, L- and D-isomers) scaffolds where one catecholate moiety of Enterobactin houses an alkene, aldehyde, or carboxylic acid at the C5 position are described. These molecules are key precursors to a family of 10 Enterobactin-cargo conjugates presented in this work, which were designed to probe the extent to which the Gram-negative ferric Enterobactin uptake and processing machinery recognizes, transports, and utilizes derivatized Enterobactin scaffolds. A series of growth recovery assays employing Enterobactin-deficient E. coli ATCC 33475 (ent-) revealed that six conjugates based on L-Ent having relatively small cargos promoted E. coli growth under iron-limiting conditions whereas negligible-to-no growth recovery was observed for four conjugates with relatively large cargos. No growth recovery was observed for the Enterobactin receptor-deficient strain of E. coli H1187 (fepA-) or the Enterobactin esterase-deficient derivative of E. coli K-12 JW0576 (fes-), or when the D-isomer of Enterobactin was employed. These results demonstrate that the E. coli ferric Enterobactin transport machinery identifies and delivers select cargo-modified scaffolds to the E. coli cytoplasm. Pseudomonas aeruginosa PAO1 K648 (pvd-, pch-) exhibited greater promiscuity than that of E. coli for the uptake and utilization of the Enterobactin-cargo conjugates, and growth promotion was observed for eight conjugates under iron-limiting conditions. Enterobactin may be utilized for delivering molecular cargos via its transport machinery to the cytoplasm of E. coli and P. aeruginosa thereby providing a means to overcome the Gram-negative outer membrane permeability barrier.
Alison Butler - One of the best experts on this subject based on the ideXlab platform.
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amphi Enterobactin commonly produced among vibrio campbellii and vibrio harveyi strains can be taken up by a novel outer membrane protein fapa that also can transport canonical fe iii Enterobactin
2018Co-Authors: Hiroaki Naka, Zachary L Reitz, Aneta L Jelowicki, Alison Butler, Margo G HaygoodAbstract:Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-Enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-Enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-Enterobactin as well as exogenous siderophores, including Enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-Enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-Enterobactins with various fatty acid appendages, including several novel amphi-Enterobactins, and these amphi-Enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-Enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-Enterobactin gene cluster, is essential for both anguibactin and amphi-Enterobactin biosynthesis, suggest the possibility that amphi-Enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.
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biosynthesis of amphi Enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional nonribosomal peptide synthetase condensation domain
2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for Enterobactin, yet Enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative Enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the Enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic Enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-Enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-Enterobactins. The amphi-Enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.
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biosynthesis of amphi Enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional nonribosomal peptide synthetase condensation domain
2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA–F) resembling that for Enterobactin, yet Enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative Enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the Enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic Enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-Enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-l-serine and acyl-l-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-Enterobactins. The amphi-Enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli a...
Hiroaki Naka - One of the best experts on this subject based on the ideXlab platform.
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amphi Enterobactin commonly produced among vibrio campbellii and vibrio harveyi strains can be taken up by a novel outer membrane protein fapa that also can transport canonical fe iii Enterobactin
2018Co-Authors: Hiroaki Naka, Zachary L Reitz, Aneta L Jelowicki, Alison Butler, Margo G HaygoodAbstract:Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-Enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-Enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-Enterobactin as well as exogenous siderophores, including Enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-Enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-Enterobactins with various fatty acid appendages, including several novel amphi-Enterobactins, and these amphi-Enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-Enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-Enterobactin gene cluster, is essential for both anguibactin and amphi-Enterobactin biosynthesis, suggest the possibility that amphi-Enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.
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biosynthesis of amphi Enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional nonribosomal peptide synthetase condensation domain
2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA-F) resembling that for Enterobactin, yet Enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative Enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the Enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic Enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-Enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-Enterobactins. The amphi-Enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli and reconstituted in vitro, demonstrating the condensation domain of AebF has unique activity, catalyzing two distinct condensation reactions.
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biosynthesis of amphi Enterobactin siderophores by vibrio harveyi baa 1116 identification of a bifunctional nonribosomal peptide synthetase condensation domain
2014Co-Authors: Hannah K Zane, Hiroaki Naka, Margo G Haygood, Federico Rosconi, Moriah Sandy, Alison ButlerAbstract:The genome of Vibrio harveyi BAA-1116 contains a nonribosomal peptide synthetase (NRPS) gene cluster (aebA–F) resembling that for Enterobactin, yet Enterobactin is not produced. A gene predicted to encode a long-chain fatty acid CoA ligase (FACL), similar to enzymes involved in the biosynthesis of acyl peptides, resides 15 kb away from the putative Enterobactin-like biosynthetic gene cluster (aebG). The proximity of this FACL gene to the Enterobactin-like synthetase suggested that V. harveyi may produce amphiphilic Enterobactin-like siderophores. Extraction of the bacterial cell pellet of V. harveyi led to the isolation and structure determination of a suite of eight amphi-Enterobactin siderophores composed of the cyclic lactone of tris-2,3-dihydroxybenzoyl-l-serine and acyl-l-serine. The FACL knockout mutant, ΔaebG V. harveyi, and the NRPS knockout mutant, ΔaebF V. harveyi, do not produce amphi-Enterobactins. The amphi-Enterobactin biosynthetic machinery was heterologously expressed in Escherichia coli a...
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identification and characterization of a novel outer membrane protein receptor feta for ferric Enterobactin transport in vibrio anguillarum 775 pjm1
2012Co-Authors: Hiroaki Naka, Jorge H CrosaAbstract:In this work we demonstrate the existence in Vibrio anguillarum 775 (pJM1) of two chromosomal genes encoding outer membrane proteins that operate in the transport of ferric Enterobactin. One of them is a novel receptor that we named FetA and the other is the already characterized FvtA that functions in the uptake of iron complexes of both Enterobactin and vanchrobactin. Ferric Enterobactin transport proficiency was resumed in double mutants for these two genes when they were complemented with either fetA or fvtA, whereas only the cloned fvtA could complement for ferric vanchrobactin transport. Quantitative RT-PCR assays demonstrated that transcription of the fetA gene is regulated by FetR, that is encoded upstream and in reverse orientation from fetA. This gene as well as fetA, are up-regulated in iron limiting condition in a Fur-dependent manner. The two divergent promoters are located in the intergenic region between fetR and fetA that has a putative Fur binding site and an IrgB binding site in the overlapping promoters of fetR and fetA. FetA and FetR show high homology to V. cholerae IrgA and IrgB respectively and the intergenic regions fetA–fetR and irgA–irgB are also highly related suggesting a vertical transmission of the fetA–fetR cluster from V. cholerae to V. anguillarum.
Phillip E. Klebba - One of the best experts on this subject based on the ideXlab platform.
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Linkage between Catecholate Siderophores and the Multicopper Oxidase CueO in Escherichia coli
2004Co-Authors: Gregor Grass, Keshari M. Thakali, Phillip E. Klebba, Daniel Thieme, Axel Müller, Günter F. Wildner, Christopher RensingAbstract:The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing Enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of Enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of Enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or Enterobactin was able to induce a Φ(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.
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three paradoxes of ferric Enterobactin uptake
2003Co-Authors: Phillip E. KlebbaAbstract:Bacteria elaborate iron chelators that scavenge iron from the environment, including their human and animal hosts, and iron acquisition is a determinant of pathogenicity. One such iron chelate, the siderophore ferric Enterobactin, enters Gram-negative bacteria through the FepA protein of the outer membrane. The ferric Enterobactin transport process is a high-affinity, multi-specific, multi-component, energy dependent reaction, that is a paradigm of ligand-gated transport: FeEnt binding activates FepA to transport competency. On the basis of the FepA, FhuA, FecA and BtuB crystal structures, and in light of recent molecular biological, biochemical, and biophysical findings, this review considers the mechanism of ferric Enterobactin uptake. The discussion focuses on three preeminent questions about the transport reaction: the function of the N-terminal globular domain that resides within the FepA channel, the mechanistic contributions of TonB to the activities of ligand-gated porins, and the energy dependence of metal transport reactions through the OM bilayer. Available data points to the idea that the N-terminal globular domains of these receptor proteins dynamically exit their pores during transport, creating a suction-force that pulls ligands through the surface loops into the periplasm. The functions of TonB and energy in these processes remain unknown.
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binding of ferric Enterobactin by the escherichia coli periplasmic protein fepb
2000Co-Authors: Cathy Sprencel, Salete M C Newton, Marjorie A Montague, Zhenghua Cao, Daniel C Scott, Nora Ivanoff, Kenneth M Raymond, Phillip E. KlebbaAbstract:The periplasmic protein FepB of Escherichia coli is a component of the ferric Enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric Enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the Kd of the ferric Enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric Enterobactin (Kd = 30 nM) and ferric enantioEnterobactin (Kd = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, Enterobactin, with comparable affinity (Kd = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric Enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.
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effect of loop deletions on the binding and transport of ferric Enterobactin by fepa
1999Co-Authors: Salete M C Newton, Daniel C Scott, John D Igo, Phillip E. KlebbaAbstract:The siderophore ferric Enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric Enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric Enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric Enterobactin transport occurred without them, at levels that allowed bacterial growth.
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selectivity of ferric Enterobactin binding and cooperativity of transport in gram negative bacteria
1998Co-Authors: Padmamalini Thulasiraman, Kenneth N Raymond, Salete M C Newton, Christine Mai, Angela Hall, Marjorie A Montague, Phillip E. KlebbaAbstract:The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric Enterobactin. We characterized the ability of seven analogs of Enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59 Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioEnterobactin, the synthetic, left-handed isomer of natural Enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric Enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd ’ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric Enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric Enterobactin with high affinity (Kd 50 pmol/min/10 9 cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric Enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.
Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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investigations of the mceij catalyzed posttranslational modification of the microcin e492 c terminus linkage of ribosomal and nonribosomal peptides to form trojan horse antibiotics
2008Co-Authors: Elizabeth M Nolan, Christopher T WalshAbstract:MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of Enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide syntheta...
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biosynthetic tailoring of microcin e492m post translational modification affords an antibacterial siderophore peptide conjugate
2007Co-Authors: Elizabeth M Nolan, Michael A Fischbach, A Koglin, Christopher T WalshAbstract:The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring Gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of Enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize Enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl)serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated Enterobactins to the C-terminal serine residue of both a C10 model peptide and full-length MccE492. In the enzymatic product, the C-terminal serine residue is covalently attached to the C4‘ oxygen of the glucose moiety. Nonenzymatic and base-cat...
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Directed evolution of aryl carrier proteins in the Enterobactin synthetase
2007Co-Authors: Zhe Zhou, Jonathan R. Lai, Christopher T WalshAbstract:The recognition of carrier proteins by multiple catalytic partners occurs in every cycle of chain elongation in the biosynthesis of fatty acids and of the pharmacologically important polyketide and nonribosomal peptide natural products. To dissect the features of carrier proteins that determine specific recognition at distinct points in assembly lines, we have used the two-module Escherichia coli Enterobactin synthetase as a model system. Using an entB knockout strain, we developed a selection for growth on iron-limiting medium to evolve aryl carrier protein domains. The aryl carrier proteins from VibB of Vibrio cholerae vibriobactin and HMWP2 of Yersinia pestis yersiniabactin assembly lines were evolved by random mutagenesis to support growth under selection conditions, yielding a convergent set of mutations. Subsequent in vitro biochemical characterizations with partner enzymes EntE, EntF, and Sfp on the evolved VibB aryl carrier protein revealed a ≈500-fold improvement in reconstituted Enterobactin production activity. Mechanistic characterization identified three distinct specific recognition surfaces of VibBArCP for three catalytic partners in Enterobactin biosynthesis. Our results suggest that heterologous carrier protein interactions can be engineered with a small number of mutations given a suitable selection scheme and provide insights for reprogramming nonribosomal peptide biosynthesis.
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in vitro characterization of salmochelin and Enterobactin trilactone hydrolases irod iroe and fes
2005Co-Authors: Hening Lin, Michael A Fischbach, David R Liu, Christopher T WalshAbstract:The iroA locus encodes five genes (iroB, iroC, iroD, iroE, iroN) that are found in pathogenic Salmonella and Escherichia coli strains. We recently reported that IroB is an Enterobactin (Ent) C-glucosyltransferase, converting the siderophore into mono-, di-, and triglucosyl Enterobactins (MGE, DGE, and TGE, respectively). Here, we report the characterization of IroD and IroE as esterases for the apo and Fe(3+)-bound forms of Ent, MGE, DGE, and TGE, and we compare their activities with those of Fes, the previously characterized Enterobactin esterase. IroD hydrolyzes both apo and Fe(3+)-bound siderophores distributively to generate DHB-Ser and/or Glc-DHB-Ser, with higher catalytic efficiencies (k(cat)/K(m)) on Fe(3+)-bound forms, suggesting that IroD is the ferric MGE/DGE esterase responsible for cytoplasmic iron release. Similarly, Fes hydrolyzes ferric Ent more efficiently than apo Ent, confirming Fes is the ferric Ent esterase responsible for Fe(3+) release from ferric Ent. Although each enzyme exhibits lower k(cat)'s processing ferric siderophores, dramatic decreases in K(m)'s for ferric siderophores result in increased catalytic efficiencies. The inability of Fes to efficiently hydrolyze ferric MGE, ferric DGE, or ferric TGE explains the requirement for IroD in the iroA cluster. IroE, in contrast, prefers apo siderophores as substrates and tends to hydrolyze the trilactone just once to produce linearized trimers. These data and the periplasmic location of IroE suggest that it hydrolyzes apo Enterobactins while they are being exported. IroD hydrolyzes apo MGE (and DGE) regioselectively to give a single linear trimer product and a single linear dimer product as determined by NMR.
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assembly line enzymology by multimodular nonribosomal peptide synthetases the thioesterase domain of e coli entf catalyzes both elongation and cyclolactonization
1999Co-Authors: Cathryn A Shawreid, Amy M Gehring, Neil L Kelleher, Heather C Losey, Christian Berg, Christopher T WalshAbstract:Background EntF is a 142 kDa four domain (condensation-adenylationpeptidyl carrier protein-thioesterase) nonribosomal peptide synthetase (NRPS) enzyme that assembles the Escherichia coli N -acyl-serine trilactone siderophore Enterobactin from serine, dihydroxybenzoate (DHB) and ATP with three other enzymes (EntB, EntD and EntE). To assess how EntF forms three ester linkages and cyclotrimerizes the covalent acyl enzyme DHB-Ser-S-PCP (peptidyl carrier protein) intermediate, we mutated residues of the proposed catalytic Ser-His-Asp triad of the thioesterase (TE) domain. Results The Ser1138→Cys mutant (k cat decreased 1000-fold compared with wild-type EntF) releases both Enterobactin (75%) and linear (DHB-Ser) 2 dimer (25%) as products. The HiResultThe Ser1138→Cys mutant (k cat decreased 1000-fold compared with wild-type EntF) releases both Enterobactin (75%) and linear (DHB-Ser) 2 dimer (25%) as products. The His1271→Ala mutant (k cat decreased 10,000-fold compared with wild-type EntF) releases only Enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser) 2 -O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates.71→Ala mutant (k cat decreased 10,000-fold compared with wild-type EntF) releases only Enterobactin, but accumulates both DHB-Ser-O-TE and (DHB-Ser) 2 -O-TE acyl enzyme intermediates. Electrospray ionization and Fourier transform mass spectrometry of proteolytic digests were used to analyze the intermediates. Conclusions These results establish that the TE domain of EntF is both a cyclotrimerizing lactone synthetase and an elongation catalyst for ester-bond formation between covalently tethered DHB-Ser moieties, a new function for chain-termination TE domains found at the carboxyl termini of multimodular NRPSs and polyketide synthases.