2 research outputs found
Mass Isotopomer Analysis of Nucleosides Isolated from RNA and DNA Using GC/MS
Nucleosides are biosynthesized from
metabolites that are at key
nodes of intermediary metabolism. Therefore, <sup>13</sup>C labeling
patterns in nucleosides from ribonucleic acid (RNA) and deoxyribonucleic
acid (DNA) in suitably designed isotopic tracer studies provide information
on metabolic flux distributions of proliferating cells. Here, we present
a gas chromatography (GC)-mass spectrometry (MS)-based approach that
permits one to exploit that potential. In order to elucidate positional
isotopomers of nucleosides from RNA and DNA, we screened the fragmentation
spectra of their trimethylsilyl derivatives. We identified the molecular
ion moieties retained in the respective fragment ions, focusing particularly
on the carbon backbone. Nucleosides fragmented at the <i>N</i>-glycosidic bond provide nucleobase and/or ribose or 2′-deoxyribose
fragment ions and fragments thereof. Nucleoside fragments composed
of the nucleobase plus some carbons of the ribose ring were also observed.
In total, we unequivocally assigned 31 fragments. The mass-isotopic
distribution of the assigned fragments provides valuable information
for later <sup>13</sup>C metabolic flux analysis as indicated by a
labeling experiment applying [1-<sup>13</sup>C]Âglucose in a yeast
culture
Biochemical and Biophysical Analysis of a Chiral PqsD Inhibitor Revealing Tight-binding Behavior and Enantiomers with Contrary Thermodynamic Signatures
Antivirulence strategies addressing
bacterial pathogenicity without
exhibiting growth inhibition effects represent a novel approach to
overcome today’s crisis in antibiotic development. In recent
studies, we examined various inhibitors of PqsD, an enzyme involved
in formation of <i>Pseudomonas aeruginosa</i> cell-to-cell
signaling molecules, and observed desired cellular effects for 2-nitrophenyl
derivatives. Herein, we investigated the binding characteristics of
this interesting compound class using several biochemical and biophysical
methods. The inhibitors showed time-dependent activity, tight-binding
behavior, and interactions with the catalytic center. Furthermore,
isothermal titration calorimetry (ITC) experiments with separated
enantiomers revealed contrary thermodynamic signatures showing either
enthalpy- or entropy-driven affinity. A combination of site-directed
mutagenesis and thermodynamic profiling was used to identify key residues
involved in inhibitor binding. This information allowed the proposal
of experimentally confirmed docking poses. Although originally designed
as transition state analogs, our results suggest an altered position
for both enantiomers. Interestingly, the main difference between stereoisomers
was found in the orientation of the hydroxyl group at the stereogenic
center. The predicted binding modes are in accordance with experimental
data and, thus, allow future structure-guided optimization