A fast screening method for the identification of siderophores from fluorescentPseudomonas spp. by liquid chromatography/electrospray mass spectrometry

Kilz, S.; Lenz, Ch.; Fuchs, Regine; Budzikiewicz, H.

doi:10.1002/(sici)1096-9888(199904)34:4<281::aid-jms750>3.0.co;2-m

Cited by 56 publications

(38 citation statements)

References 42 publications

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“…Although the C-7 position of the chromophore is most commonly a hydride, sulfonyl and chloride groups have also been observed experimentally (8). Finally, pyoverdines isolated from P. aeruginosa have been observed to contain a single or double bond between the C-5 and C-6 position of the chromophore (9).…”

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confidence: 99%

“…The pyoverdine molecule contains proteinogenic amino acids as well as the unusual amino acids 2,4-diaminobutyrate and formyl-N-hydroxyornithine. P. aeruginosa produces multiple isoforms of pyoverdine that have been identified by mass spectrometry (8,9). The peptide backbone, as dictated by the NRPS enzymes, remains constant, but variations in the chromophore and the N-terminal side chain have been observed.…”

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confidence: 99%

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The 1.8 Å Crystal Structure of PA2412, an MbtH-like Protein from the Pyoverdine Cluster of Pseudomonas aeruginosa

Drake

Cao

et al. 2007

Journal of Biological Chemistry

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Many bacteria use nonribosomal peptide synthetase (NRPS) proteins to produce peptide antibiotics and siderophores. The catalytic domains of the NRPS proteins are usually linked in large multidomain proteins. Often, additional proteins are coexpressed with NRPS proteins that modify the NRPS peptide products, ensure the availability of substrate building blocks, or play a role in the import or export of the NRPS product. Many NRPS clusters include a small protein of ϳ80 amino acids with homology to the MbtH protein of mycobactin synthesis in Mycobacteria tuberculosis; no function has been assigned to these proteins. Pseudomonas aeruginosa utilizes an NRPS cluster to synthesize the siderophore pyoverdine. The pyoverdine peptide contains a dihydroxyquinoline-based chromophore, as well as two formyl-N-hydroxyornithine residues, which are involved in iron binding. The pyoverdine cluster contains four modular NRPS enzymes and 10 -15 additional proteins that are essential for pyoverdine production. Coexpressed with the pyoverdine synthetic enzymes is a 72-amino acid MbtH-like family member designated PA2412. We have determined the three-dimensional structure of the PA2412 protein and describe here the structure and the location of conserved regions. Additionally, we have further analyzed a deletion mutant of the PA2412 protein for growth and pyoverdine production. Our results demonstrate that PA2412 is necessary for the production or secretion of pyoverdine at normal levels. The PA2412 deletion strain is able to use exogenously produced pyoverdine, showing that there is no defect in the uptake or utilization of the iron-pyoverdine complex.Iron is an essential cofactor for many proteins, playing both catalytic and structural roles (1-3). Because of the low solubility of free Fe 3ϩ , many bacteria produce siderophores that bind to iron. The iron-siderophore complex is then actively transported into the cell. Although some small molecules like citrate or salicylate are able to function as lower affinity siderophores, many specialized peptides with extremely high affinity for iron are produced nonribosomally by bacteria. These compounds are produced by nonribosomal peptide synthetases (NRPSs) 2 that exist as modular proteins with multiple catalytic domains joined in a single protein. Often expressed with NRPS genes are other genes that encode proteins involved in additional essential steps including the synthesis of building blocks, siderophore export, import of the Fe 3ϩ -siderophore complex, or the removal Fe 3ϩ from the imported siderophore (4, 5). Pseudomonas aeruginosa is a bacterial pathogen that commonly causes infections in patients with cystic fibrosis (6). The siderophore pyoverdine (Fig. 1) is synthesized by P. aeruginosa (3) and has been correlated with virulence (7). Pyoverdine contains a cyclic peptide chain as well as a chemically modified dihydroxyquinoline-based chromophore that is responsible for iron binding. The peptide backbone of pyoverdine is synthesized by four large, multi-module NRPS proteins. The ...

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The 1.8 Å Crystal Structure of PA2412, an MbtH-like Protein from the Pyoverdine Cluster of Pseudomonas aeruginosa

Drake

Cao

et al. 2007

Journal of Biological Chemistry

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“…Electrospray ionization mass spectrometry (ESI MS) and tandem mass spectrometry (MS/MS) have proved to be powerful techniques for detecting and identifying siderophores [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23]. For example, Gledhill demonstrated the utility of these techniques for characterizing hydroxamate-type siderophores, including ferrioxamine, ferrichrome, and iron(III) rhodotoluate [9].…”

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confidence: 99%

Collision-activated dissociation, infrared multiphoton dissociation, and electron capture dissociation of the Bacillus anthracis siderophore petrobactin and its metal ion complexes

Liu

Håkansson

Lee

et al. 2007

J. Am. Soc. Mass Spectrom.

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Siderophores are high-affinity iron-chelating ligands produced by microorganisms to scavenge vital Fe 3ϩ from the environment. Thus, siderophores constitute potential therapeutic targets and their structural determination is important for exploiting their therapeutic value. Here, the virulence-associated siderophore petrobactin from Bacillus anthracis was characterized with electron capture dissociation (ECD). Fragmentation of doubly protonated petrobactin was investigated and compared to sustained off-resonance irradiation collision-activated dissociation (SORI CAD) and infrared multiphoton dissociation (IRMPD) of both the singly and doubly protonated species. These experiments demonstrate that ECD provides additional information (complementary bond cleavages) on the structure of petrobactin compared to both SORI CAD and IRMPD. Furthermore, complexes of petrobactin with divalent (Ca , and Co 2ϩ ) and trivalent (Fe 3ϩ and Ga 3ϩ ) metal cations were also subjected to SORI CAD and ECD. Again, most structural information was obtained from the ECD spectra. However, significant differences were found in both SORI CAD and ECD of metal complexes, dependent on the nature of the metal ion. Intriguingly, unique behavior, consistent with a recently proposed solution-phase structure, was observed for the highly preferred Fe 3ϩ -petrobactin complex. (J Am Soc Mass Spectrom 2007, 18, 842-849)

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“…It is known that bacterial lipopeptides and cyclic peptides, including pyoverdines, are generally produced as a mixture with different modifications such as hydroxylation, amidation of carboxylic acid, and differ-ent alkyl chain lengths in the fatty acid part. 16,19) The fact that all the peaks were eluted under the 10% acetonitrile condition on the HPLC suggested that Pvd38 contains several hydrophilic amino acids in the peptide part.…”

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confidence: 95%

“…Before injection into HPLC, culture supernatant containing pyoverdine was roughly purified using a Sep-Pak RP-C 18 cartridge (Waters, Milford, MA). 16) After the cartridge was washed with water, the fraction containing pyoverdine was recovered at 70% methanol and then diluted with water to 5% methanol solution. Insoluble compounds were removed by centrifuge (8;000 g for 10 min), and the resulting clear solution was applied to HPLC.…”

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confidence: 99%

Functional Analysis of A Pyoverdine Synthetase fromPseudomonassp. MIS38

Lim

Roongsawang

Washio

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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A fast screening method for the identification of siderophores from fluorescentPseudomonas spp. by liquid chromatography/electrospray mass spectrometry

Cited by 56 publications

References 42 publications

The 1.8 Å Crystal Structure of PA2412, an MbtH-like Protein from the Pyoverdine Cluster of Pseudomonas aeruginosa

The 1.8 Å Crystal Structure of PA2412, an MbtH-like Protein from the Pyoverdine Cluster of Pseudomonas aeruginosa

Collision-activated dissociation, infrared multiphoton dissociation, and electron capture dissociation of the Bacillus anthracis siderophore petrobactin and its metal ion complexes

Functional Analysis of A Pyoverdine Synthetase fromPseudomonassp. MIS38

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