Stefan Freund

Purification and chemical characterization of staphyloferrin B, a hydrophilic siderophore from staphylococci

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicus DSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 mHCl revealed the presence of l-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.

Rhizoferrin — a novel siderophore from the fungusRhizopus microsporus var.rhizopodiformis

SummaryFrom a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN1,N4-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.

Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn1(Nδ-OH, NΔ-acyl)-d-threo-Asp(β-OH)-l-Ser-l-Orn4(Nδ-OH, Nδ-formyl)-1,4-diaminobutane. The Nδ-acyl groups of Orn1(Nδ-OH, Nδ-acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.

Lantibiotics : An Overview and Conformational Studies on Gallidermin and Pep5

Lantibiotics are polycyclic bacteriocins with the thioether amino acids lanthionine and β - methyllanthionine. In addition to these intrachain sulfide bridges, unsaturated amino acids like α,β- didehydroalanine and α, β - didehydrobutyric acid occur. Members of the lantibiotics family are for example nisin, an important food preservative, epidermin, a highly specific therapeutic drug against acne, a series of enzyme inhibitors and immunologically interesting peptides such as the duramycins. In recent years, an increasing number of novel lantibiotics has been discovered. Lantibiotics are produced via ribosomal synthesis from inactive precursor proteins (prelantibiotics), which are post-translationally converted to the biologically active polycyclic peptides. The enzyme involved herein catalyzes the dehydration of the serine and threonine residues to yield unsaturated residues. This step is followed by a stereospecific addition of the cysteine thiol groups to these double bonds leading to the formation of sulfide bridges. The final step is the release of the active peptide by proteolytic cleavage of the leader peptide. Conformational analysis of the lantibiotics, as well as their prepeptides, provide information about their mode of action and the steps of biosynthesis. Based on the knowledge of the lantibiotic biosynthesis, and by using the isolated novel enzymes, a gene technological construction of a variety of similarly modified polypeptides will be available in the near future.

Ubiquitin Binds to a Short Peptide Segment of Hydrolase UCH‐L3: A Study by FCS, RIfS, ITC and NMR

Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL‐NH2 as a specific ubiquitin‐interacting ligand. A peptide collection—based on crystal structures with ubiquitin‐interacting proteins—was designed and confirmed by sequence comparison of ubiquitin‐interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 μM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface‐immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. 1H,15N heteronuclear NMR localised the interaction site across the β sheet of ubiquitin. The peptide aligns well with the ubiquitin‐interacting motif and represents a lead structure for the rational design of high‐affinity tags for targeting ubiquitinated protein in vitro and in vivo.