Fr Francium

The inside cover picture shows fluorescently labeled β-tubulin in live cells. β-Tubulin is expressed as a SNAP-tag fusion and site-specifically labeled with fluorogenic benzylguanine probes. These intramolecularly quenched probes are fluorescent only after reaction with SNAP-tag, thus eliminating the need for washing steps when visualizing cellular targets. For further details on the characterization and application of these probes, see the paper by I. R. Corrêa, Jr. et al. on p. 2217 ff.

The cover picture shows a schematic structure of Aculeine A, a novel peptide toxin modified with long-chain polyamines (LCPAs), and the marine sponge Axynissa acureata, from which the peptide was isolated. In the background is an underwater photograph taken off Iriomote Island, Okinawa (Japan) where this sponge is found. Aculeines, which induce convulsions in mice upon intra-cerebroventricular injection and disrupt neuronal membrane integrity in electrophysiological assays, are 44-residue ribosomal peptides whose amino acid sequence contains a cysteine-knot motif. The N-terminal residue is a highly modified tryptophan that is associated with polypropanamine oligomers of up to 15 repetitive units. The highly modified N-terminal structure is an unprecedented structural feature among naturally occurring peptides. For more information, see the paper by R. Sakai et al. on p. 2191 ff.

Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.

Antibiotic biosynthesis: Thienamycin and (5R)-carbapen-2-em-3-carboxylate (1) are structurally related carbapenem β-lactam antibiotics that are produced by phylogenetically distant organisms. The biosynthesis of 1 is well understood, thienamycin biosynthesis is not. Here we demonstrate that formation of these carbapenems is functionally and stereochemically identical through only the first two biosynthetic steps.

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3′ and 5′ RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.

Armed with LCPA: Aculeines (ACUs), isolated from the marine sponge Axinyssa aculeata are novel polypeptide toxins modified with long-chain polyamine (LCPA). We show here that ACU-A and B share a 45-residue ribosomal polypeptide but are modified differently at the N-terminal tryptophan. ACU-A exhibits hemolytic, cytotoxic, and seizurogenic activity in mice, and was shown to disrupt plasma membranes.

Looped in: Modular assembly provides a unique opportunity to tune the affinity and specificity of ligands for a target biomolecule. Herein, we describe our investigations into how the distance between bis-benzimidazole ligand modules (see graphic) affects affinity and specificity for RNA internal loops.

Cellular proteins that bear reactive azides can be imaged by fluorescence microscopy following strain-promoted ligation to cyclooctyne dyes. Here we describe BODIPY-cyclooctyne (BDPY), a membrane-permeant fluorophore that can be used to label intracellular proteins in live mammalian cells. Flow cytometry reveals fluorescence signals more than 25-fold above background after labeling of azide-tagged cells with BDPY.