MgF and AlF transition state analogue complexes of yeast phosphoglycerate kinase.

The phospho-transfer mechanism of yeast phosphoglycerate kinase (PGK) has been probed through formation of trifluoromagnesate (MgF) and tetrafluoroaluminate (AlF) transition state analogue complexes and analyzed using F, H waterLOGSY and H chemical shift perturbation NMR spectroscopy. We observed the first F NMR spectroscopic evidence for the formation of metal fluoride transition state analogues of yeast PGK and also observed significant changes to proton chemical shifts of PGK in the presence, but not in the absence, of fluoride upon titration of ligands, providing indirect evidence of the formation of a closed ternary transition state. WaterLOGSY NMR spectroscopy experiments using an uncompetitive model were used in an attempt to measure ligand binding affinities within the transition state analogue complexes.

JadX is a Disparate Natural Product Binding Protein.

We report that JadX, a protein of previously undetermined function coded for in the jadomycin biosynthetic gene cluster of Streptomyces venezuelae ISP5230, affects both chloramphenicol and jadomycin production levels in blocked mutants. Characterization of recombinant JadX through protein-ligand interactions by chemical shift perturbation and WaterLOGSY NMR spectroscopy resulted in the observation of binding between JadX and a series of jadomycins and between JadX and chloramphenicol, another natural product produced by S. venezuelae ISP5230.

Characterization of l-Digitoxosyl-phenanthroviridin from Streptomyces venezuelae ISP5230.

The jadomycin-derived compound l-digitoxosyl-phenanthroviridin was isolated from fermentations of Streptomyces venezuelae ISP5230 grown in nutrient-deficient media with l-lysine as the sole nitrogen source. Structural elucidation was accomplished using a combination of high-resolution MS, LC-MS/MS, and 1D- and 2D-NMR. The compound was evaluated against the National Cancer Institute (NCI) 60 human tumor cell line screen in both the one-dose and five-dose screens, and cytotoxicity was compared to a small library of jadomycin analogues to probe the structure-activity relationship.

Isolation and Synthetic Diversification of Jadomycin 4-Amino-l-phenylalanine.

Streptomyces venezuelae ISP5230 was grown in the presence of phenylalanine analogues to observe whether they could be incorporated into novel jadomycin structures. It was found that the bacteria successfully produced jadomycins incorporating 4-aminophenylalanine enantiomers. Upon isolation and characterization of jadomycin 4-amino-l-phenylalanine (1), it was synthetically derivatized, using activated succinimidyl esters, to yield a small jadomycin amide library.

Eight-membered ring-containing jadomycins: implications for non-enzymatic natural products biosynthesis.

Jadomycin Oct (1) was isolated from Streptomyces venezuelae ISP5230 and characterized as a structurally unique eight-membered l-ornithine ring-containing jadomycin. The structure was elucidated through the semisynthetic derivatization of starting material via chemoselective acylation of the l-ornithine α-amino group using activated succinimidyl esters. Incorporation of 5-aminovaleric acid led to jadomycin AVA, a second eight-membered ring-containing jadomycin.

Polyphosphate-containing bisubstrate analogues as inhibitors of a bacterial cell wall thymidylyltransferase.

A series of polyphosphate containing sugar nucleotide analogues were synthesized and evaluated as bisubstrate inhibitors of α-D-glucose 1-phosphate thymidylyltransferase Cps2L, the first enzyme in Streptococcus pneumoniael-rhamnose biosynthesis, and a novel antibacterial target. WaterLOGSY NMR spectroscopy demonstrated binding of bisubstrate analogues to Cps2L and a spectrophotometric coupled assay was used to determine apparent Ki values.

In vivo detection of human TRPV6-rich tumors with anti-cancer peptides derived from soricidin.

Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors.

Cell-cell membrane fusion induced by p15 fusion-associated small transmembrane (FAST) protein requires a novel fusion peptide motif containing a myristoylated polyproline type II helix.

The p15 fusion-associated small transmembrane (FAST) protein is a nonstructural viral protein that induces cell-cell fusion and syncytium formation. The exceptionally small, myristoylated N-terminal ectodomain of p15 lacks any of the defining features of a typical viral fusion protein. NMR and CD spectroscopy indicate this small fusion module comprises a left-handed polyproline type II (PPII) helix flanked by small, unstructured N and C termini.

Jadomycins derived from the assimilation and incorporation of norvaline and norleucine.

Streptomyces venezuelae ISP5230 is recognized for the production of chloramphenicol and the jadomycin family of natural products. The jadomycins are angucycline natural products containing a unique oxazolone ring incorporating an amino acid present in the minimal culture media. Substitution of different amino acids results in products of varying biological activity.

A method for structure-activity analysis of quorum-sensing signaling peptides from naturally transformable streptococci.

Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure-function relationships.

Structure-activity analysis of quorum-sensing signaling peptides from Streptococcus mutans.

Streptococcus mutans secretes and utilizes a 21-amino-acid signaling peptide pheromone to initiate quorum sensing for genetic competence, biofilm formation, stress responses, and bacteriocin production. In this study, we designed and synthesized a series of truncated peptides and peptides with amino acid substitutions to investigate their structure-activity relationships based on the three-dimensional structures of S. mutans wild-type signaling peptide UA159sp and C-terminally truncated peptide TPC3 from mutant JH1005 defective in genetic competence.

Structural characterization of the antimicrobial peptide pleurocidin from winter flounder.

Pleurocidin is an antimicrobial peptide that was isolated from the mucus membranes of winter flounder (Pseudopleuronectes americanus) and contributes to the initial stages of defense against bacterial infection. From NMR structural studies with the uniformly (15)N-labeled peptide, a structure of pleurocidin was determined to be in a random coil conformation in aqueous solution whereas it assumes an alpha-helical structure in TFE and in dodecylphosphocholine (DPC) micelles. From (15)N relaxation studies, the helix is a rigid structure in the membrane-mimicking environment.

Silicon-29 NMR Studies of Tetraalkylammonium Silicate Solutions. 2. Polymerization Kinetics.

The kinetics of formation of the silicate cubic octamer, Q(3)(8), in aqueous tetramethylammonium (TMA) silicate solutions was investigated by (29)Si NMR. The rate equation for solutions at pH 13.2-13.

Silicon-29 NMR Studies of Tetraalkylammonium Silicate Solutions. 1. Equilibria, (29)Si Chemical Shifts, and (29)Si Relaxation.

The addition of tetraalkylammonium cations to aqueous silicate solutions enhances the abundance of symmetric, cagelike, polysilicate anions including the cubic octamer, Si(8)O(20)(8)(-). The equilibrium ratio of tetramethylammonium (TMA) cations to the octameric silicate anion is 8:1 for solutions with a concentration ratio [OH(-)]:[Si] >/= 1:1. Evidence indicates that organocations directly associate with cagelike polyanions to form a protective shell of hydrophobic hydration that impedes hydrolysis of the central anion.