Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens.

The freshwater cyanobacterium Planktothrix rubescens produces the cyclooctapeptide cyclo(Pro-Gly-Leu-Val-Met-Phe-Gly-Val). The chemical structure is new. This homodetic cyclic octapeptide was named planktocyclin ( 1). It consists solely of proteinogenic l-amino acids and is a strong inhibitor of mammalian trypsin and alpha-chymotrypsin and a moderately active inhibitor of human recombinant caspase-8. Mass spectrometric and 2D-NMR spectroscopic data allowed the determination of its structure. Synthetic planktocyclin was identical to the natural product.

A genomic screening approach to the structure-guided identification of drug candidates from natural sources.

The potential of actinomycetes to produce natural products has been exploited for decades. Recent genomic sequence analyses have revealed a previously unrecognized biosynthetic potential and diversity. In order to rationally exploit this potential, we have developed a sequence-guided genetic screening strategy. In this "genome mining" approach, genes that encode tailoring enzymes from natural product biosyntheses pathways serve as indicator genes for the identification of strains that have the genetic potential to produce natural products of interest. We chose halogenases, which are known to be involved in the synthesis of halometabolites as representative examples. From PCR screening of 550 randomly selected actinomycetes strains, we identified 103 novel putative halogenase genes. A phylogenetic analysis of the corresponding putative halogenases, and the determination of their sequential context with mass spectrometric analysis of cultures filtrates revealed a distinct correlation between the sequence and secondary metabolite class of the halometabolite. The described screening strategy allows rapid access to novel natural products with predetermined structural properties.

Evaluation of enantioselective gas chromatography for the determination of minute deviations from racemic composition of alpha-amino acids with emphasis on tyrosine: accuracy and precision of the method.

Whereas the determination of high enantiomeric fractions (EF) of chiral compounds is very well established, the accurate determination of small deviations from racemic compositions has not yet received much attention despite its relevance to studies dealing with the origin of homochirality, where only small initial enantiomeric bias is expected. Racemic samples of representative alpha-amino acids were derivatized as N-(O,S)-trifluoroacetyl/ethylesters and analyzed by enantioselective gas chromatography (GC) on fused silica capillaries coated with the chiral stationary phases (CSPs) Chirasil-D-Val, Chirasil-L-Val, and Lipodex E with GC/FID and GC/MS detection. The validation (accuracy and precision) of the determination of the enantiomeric fraction EF of the D-enantiomer in racemic or near-racemic compositions for 10 DL-alpha-amino acids obtained from commercial sources has been carried out. Emphasis is given to DL-tyrosine, the enantiomers of which have recently been claimed to show different crystallization properties. Values of EF obtained from GC measurements using CSPs were compared with those from CE using chiral mobile phase additives. While the precision of the GC method is generally better than 0.08% for all DL-alpha-amino acids studied, accuracy (trueness) of determination of amino acids with polar side chains is poorer than expected from the precision as a result of systematic errors. The accuracy determined relied on measurements on two oppositely configurated CSPs.

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna.

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.

Parity violating energetic difference and enantiomorphous crystalsp-caveats; reinvestigation of tyrosine crystallization.

The present article challenges reports claiming to have demonstrated the Parity Violating Energetic Difference (PVED) between enantiomorphous D- and L-crystals. Apart from PVED, the presence of minute quantities and differing profiles of impurities incorporated during their different history of preparation will affect the physical properties of D- and L-crystals. These impurities are anticipated to play a much greater role in affecting crystallization behavior than PVED. The effect of impurities on the growth and dissolution of enantiomorphous crystals is illustrated with some representative examples. Shinitzky et al. (2002) reported recently dramatic differences in the growth and dissolution properties of the D- and L-crystals of tyrosine. We have repeated these experiments using commercial samples from different sources and employing a validated enantioselective gas chromatographic technique. We attribute Shinitzky's findings either to the use of inappropriate analytical techniques for the determination of enantiomeric composition and/or to the presence of unidentified contaminants in the commercial tyrosine samples. Related caveats hold also for the recently published claims by Shinitzky (2006) and Scolnik et al. (2006) to have observed experimentally PVED between enantiomeric helices of poly-glutamic acid composed of 24 repeating units.

The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica.

Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

Mutasynthesis of glycopeptide antibiotics: variations of vancomycin's AB-ring amino acid 3,5-dihydroxyphenylglycine.

In the mutasynthetic approach, the DeltadpgA mutant of the vancomycin-type glycopeptide antibiotic producer Amycolatopsis balhimycina, which is deficient in the synthesis of 3,5-dihydroxyphenylglycine (DPg), was supplemented with synthetic DPg analogues to obtain the corresponding modified glycopeptides. Sterically more demanding 3,5-disubstituted methoxy derivatives as well as monosubstituted DPg analogues were accepted as substrates. These facts indicate that steric and electronic requirements suffice in several cases for the oxidative closure of the AB ring, thus leading to the generation of novel antibiotically active glycopeptide derivatives. The results represent a further step in evaluating the potential of mutasynthesis for peptidic secondary metabolites.

Aspochalamins A-D and aspochalasin Z produced by the endosymbiotic Fungus Aspergillus niveus LU 9575. II. Structure elucidation.

The structures of new cytochalasan fungal metabolites aspochalamins A-D have been elucidated by ESI-FTICR-MS, NMR spectroscopy, and chiral amino acid analysis. Aspochalamins A-D consist of different aspochalasin skeletons connected at position C-19 to the N terminus of the tripeptidic moiety amide anthranoyl-L-alanine-E-didehydrotryptamide. Furthermore, the structure of a new aspochalasin analog, aspochalasin Z, was derived from its molecular mass and NMR data as 10-isopropyl-14-methyl[11]-cytochalasa-6Z,13E,19E-triene-1,21-dione.

Arylomycins A and B, new biaryl-bridged lipopeptide antibiotics produced by Streptomyces sp. Tü 6075. II. Structure elucidation.

The structures of new lipopeptide antibiotics, arylomycins A and B, were elucidated by a combination of ESI-FTICR-mass spectrometry, NMR spectroscopy, Edman sequencing, and fatty acid and chiral amino acid analyses. The colourless arylomycins A share the peptide sequence of D-N-methylseryl2(D-MeSer2)-D-alanyl3-glycyl4-N-methyl- 4-hydroxyphenylglycyl5(MeHpg5)-L-alanyl6-tyrosine7 cyclised by a [3,3]biaryl bond between MeHpg5 and Tyr7. The yellow arylomycins B differ from arylomycins A by nitro substitution of Tyr7. The N-termini of arylomycins A and B are acylated with saturated C11-C15 fatty acids (fa1) comprising n, iso, and anteiso isomers. Arylomycins A and B represent the first examples of biaryl-bridged lipopeptides.

Detection of bone glue treatment as a major source of contamination in ancient DNA analyses.

Paleogenetic investigations of ancient DNA extracted from fossil material is for many reasons susceptible to falsification by the presence of more recent contamination from several sources. Gelatine-based bone glue that has been used extensively for nearly two centuries by curators to preserve hard tissues contributes nonauthentic DNA to paleontological material. This fact has been frequently neglected and is barely mentioned in the literature. Now paleogeneticists, curators, and conservators are faced with the problem that treatment of samples with adhesives and consolidants for conservatory purposes has seldom been recorded. Here, we show that racemization of amino acids, and in particular serine, is an excellent indicator for the treatment of paleontological samples with glue.