Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

Abstract

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

Elusive structural changes of New Delhi metallo-β-lactamase revealed by ultraviolet photodissociation mass spectrometry

“…† Regions of the protein in which noncovalent interactions or exibility are impacted by single point mutations can be elucidated by examining the enhancement or suppression of UVPD backbone cleavages. [35][36][37] For each NDM variant, the backbone cleavage efficiency upon UVPD was determined and is represented graphically in Fig. S20 † as a function of each residue of the protein.…”

“…31 Additionally, UVPD affords unsurpassed sequence coverage 29,30 and retention of labile covalent moieties 32 with little dependence on precursor charge state. 33 This native UVPD-MS approach has previously been applied for detecting conformational changes induced by ligand binding to dihydrofolate reductase (DHFR), 34 sequence variants of rat sarcoma GTPase K-Ras 35,36 and DHFR, 37 and structural re-organization of the phosphotransferase enzyme adenylate kinase during its catalytic cycle. 38 Here, we use native UVPD-MS to track changes in fragmentation patterns as a means to infer changes in the active site loop conformation, zinc binding, and conformations of surrounding residues in NDM-1 upon binding to three different small molecule inhibitors known to covalently modify the enzyme.…”

Chemical space of Escherichia coli dihydrofolate reductase inhibitors: New approaches for discovering novel drugs for old bugs

“…On the contrary, non-classical inhibitors can diffuse passively through cell membranes. A prominent example includes trimethoprim, which is weakly basic at physiological pH and shows potent cytotoxicity for E. coli (Its MIC is approximately 6.9 μM 76 ) in spite of showing poorer affinity (vis-à-vis methotrexate) for the recombinantly expressed bacterial enzyme (IC 50 is ~20.4 ± 2.3 nM 77 ).…”

The Applications of Promoter-gene-Engineered Biosensors

“…Therefore, the investigation of the nucleocytoplasmic shuttling mechanism of YAP enables us to deeply understand the tumorigenesis and oncology mechanism, and find more effective tumor therapy ( Figure 6 ). Furthermore, if both mCherry and EGFP gene are fused with individual interactive proteins or enzymes, we can investigate the protein–protein interactions and bacteria–host cell interactions associated with the pathogenesis of infectious diseases and the mechanism of the catalytic reaction of enzymes and their activity in the cell array [ 20 , 21 , 22 , 23 ] ( Figure 5 and Figure 7 ).…”