Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.
Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.
The conformation of microtubule-bound paclitaxel has been examined by fluorescence and solid-state NMR spectroscopy. A fluorescent derivative of paclitaxel, 3'-N-debenzoyl-3'-N-(m-aminobenzoyl)paclitaxel (N-AB-PT), was prepared by semisynthesis. No differences in the microtubule-promoting activity between N-AB-PT and paclitaxel were observed, demonstrating that addition of the amino group did not adversely affect the ligand-receptor association. The distance between the fluorophore N-AB-PT and the colchicine binding site on tubulin polymers was determined through time-resolved measurements of fluorescence resonance energy transfer to be 29 +/- 2 A. The absorption and emission spectra of N-AB-PT bound to microtubules and in various solvents were measured. A plot of the Stokes shift as a function of solvent polarity was highly unusual. The Stokes shift increased linearly with solvent polarity in protic solvents, which is expected due to the nature of the fluorophore. In aprotic solvents, however, the Stokes shift was invariant with solvent polarity, indicating that the fluorophore was somehow shielded from the effects of the solvent. These data are best explained by considering the solution-state conformational properties of paclitaxel. It is known that paclitaxel adopts different conformations depending on the nature of the solvent, and these fluorescence data are consistent with the molecule adopting a "hydrophobic collapsed" conformation in protic solvents and an "extended" conformation in aprotic solvents. The Stokes shift of microtubule-bound N-AB-PT was within the protic solvent region, demonstrating that microtubule-bound paclitaxel is in a hydrophobic collapsed conformation. Microtubule-bound paclitaxel was also investigated by solid-state NMR. Paclitaxel was labeled with (19)F at the para position of the C-2 benzoyl substituent and with (13)C and (15)N in the side chain. Distances between the fluorine and carbon nuclei were determined by REDOR. The distance between the fluorine and the 3'-amide carbonyl carbon was 9.8 +/- 0.5 A, and the distance between the fluorine atom and the 3'-methine carbon was 10. 3 +/- 0.5 A. These spectroscopic data were used in conjunction with molecular modeling to refine the microtubule-bound conformation of paclitaxel and to suggest an alternative orientation of the ligand within the paclitaxel binding site.
The 15N{19F} REDOR NMR spectra of two fluorolumazines complexed to the 1-MDa β60 capsid of lumazine synthase have been obtained at 20.3 and 50.7 MHz. Distances from CF3 groups of the ligands to six side- and main-chain nitrogens have been measured. These distances were used in combination with the X-ray crystal coordinates of wild-type lumazine synthase, complexed to a related substrate ligand, in a series of distance-restrained molecular dynamics simulations. The result is a model of the binding site of lumazine synthase that has sufficient detail to predict the absolute configuration at C-7 of complexed 7-hydroxy-8-d-ribityl-6,7-bis(trifluoromethyl)-7,8-dihydropteridine-2,4(1H,3H)-dione, a fluorinated analogue of an unstable, hypothetical intermediate in the reactions catalyzed by both lumazine synthase and riboflavin synthase.
Glycosaminoglycans (GAGs) are linear polysaccharides made by all animal cells. GAGs bind to hundreds of proteins, such as growth factors, cytokines, chemokines, extracellular matrix components, protease inhibitors, proteases, and lipoprotein lipase, through carbohydrate and protein interactions. These interactions control many multicellular processes. The increased use of GAGs isolated from cells and small tissue samples in bioassays and binding experiments demands a sensitive and robust quantification method. We have developed such a method, which is based on a popular assay for amino acid analysis. We have refined it to enhance GAG quantification. It allows the quantification of glucosamine- and galactosamine-containing GAGs after the reversed-phase separation of their fluorescent isoindole derivatives. The derivatives are created by the reaction of o-phthaldialdehyde and 3-mercaptopropionic acid (3MPA) with the amino group of hexosaminitol monosaccharides generated from GAG acid hydrolysis and sodium borohydride reduction. The advantages of our method include automatic derivitization, a simple chromatograph with clean separation of glucosaminitol and galactosaminitol derivatives from contaminating amino acids, excellent sensitivity with 0.04 pmol detection, and linearity from 2.5 to 1280 pmol. A major advantage is that it can be readily implemented in any laboratory with typical reversed-phase high performance liquid chromatography (HPLC) equipment.
EPSP (5-enolpyruvylshikimate-3-phosphate) synthase catalyzes condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor to the aromatic amino acids. S3P and [2-13C]POP were bound to mutant or wild type E. coli forms of the enzyme prior to lyophilization. CPMAS-echo and rotational-echo double-resonance (REDOR) NMR experiments, employing a slow catalytic EPSP synthase mutant and a long prelyophilization incubation interval, allowed our observation of the gradual formation of a strong 31P-13C coupling consistent with the well characterized tetrahedral intermediate. However, after shorter low temperature incubation intervals of substrates with mutant or wild-type enzymes, carbon CPMAS-echo NMR spectra showed the 13C label at 155 ppm, consistent with sp2 geometry of this carbon. REDOR revealed that the phosphorus of PEP was cleaved. However, phosphorus at a distance of 7.5 A was observed, due to the phosphate of a nearby bound S3P. Heating the sample allowed the reaction to progress, as shown by the diminution of the 155 ppm peak and growth of a peak at 108 ppm. The sp3 geometry implied by the 108 ppm peak strongly suggested formation of a S3P-PEP condensation product. REDOR indicated that phosphorus was still distant, but now only 6.1 (wild type) or 5.9 A (mutant) distant. We think that the early intermediates with peaks at 155 and 108 ppm are covalently bound to the enzyme. We also think that the tetrahedral intermediate that we observed was formed after product was generated.
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