ABSTRACT
The development of new antibiotics is necessitated by the rapid development of resistance to current therapies. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), which catalyzes the first committed step of bacterial peptidoglycan biosynthesis, is a prime candidate for therapeutic intervention. MurA is the target of the antibiotic fosfomycin, a natural product produced by Streptomyces. Despite possessing a high degree of sequence conservation with MurA enzymes from fosfomycin-susceptible organisms, recent microbiological studies suggest that MurA from Vibrio fischeri (VfiMurA) may confer fosfomycin resistance via a mechanism that is not yet understood. The crystal structure of VfiMurA in a ternary complex with the substrate UDP-N-acetylglucosamine (UNAG) and fosfomycin has been solved to a resolution of 1.93 Å. Fosfomycin is known to inhibit MurA by covalently binding to a highly conserved cysteine in the active site of the enzyme. A comparison of the title structure with the structure of fosfomycin-susceptible Haemophilus influenzae MurA (PDB entry 2rl2) revealed strikingly similar conformations of the mobile substrate-binding loop and clear electron density for a fosfomycin-cysteine adduct. Based on these results, there are no distinguishing sequence/structural features in VfiMurA that would translate to a diminished sensitivity to fosfomycin. However, VfiMurA is a robust crystallizer and shares high sequence identity with many clinically relevant bacterial pathogens. Thus, it would serve as an ideal system for use in the structure-guided optimization of new antibacterial agents.
Subject(s)
Aliivibrio fischeri/enzymology , Alkyl and Aryl Transferases/chemistry , Fosfomycin/chemistry , Protein Interaction Domains and Motifs , Uridine Diphosphate N-Acetylglucosamine/chemistry , Alkyl and Aryl Transferases/metabolism , Fosfomycin/metabolism , Models, Molecular , Substrate Specificity , Uridine Diphosphate N-Acetylglucosamine/metabolismABSTRACT
We report a series of measurements directed to assess the suitability of alanine as a mailable dosimeter for dosimetry quality assurance of proton radiation therapy beams. These measurements include dose-response of alanine at 140 MeV, and comparison of response vs energy with a parallel plate ionization chamber. All irradiations were made at the Harvard Cyclotron Laboratory, and the dosimeters were read at NIST. The results encourage us that alanine could be expected to serve as a mailable dosimeter with systematic error due to differential energy response no greater than 3% when doses of 25 Gy are used.
Subject(s)
Alanine/radiation effects , Electron Spin Resonance Spectroscopy/methods , Proton Therapy , Radiometry/methods , Radiotherapy Dosage , Alanine/chemistry , Dose-Response Relationship, Radiation , Electron Spin Resonance Spectroscopy/standards , Electron Spin Resonance Spectroscopy/statistics & numerical data , Evaluation Studies as Topic , Free Radicals/analysis , Free Radicals/radiation effects , Humans , Radiometry/instrumentation , Radiometry/standards , Radiotherapy Dosage/standards , Radiotherapy, High-EnergyABSTRACT
Cancer therapy studies using proton accelerators are underway in several major medical centers in the U.S., Russia, Japan and elsewhere. To facilitate dosimetry intercomparisons between these laboratories, alanine-based detectors produced at the National Institute of Standards and Technology and commercially available radiochromic films were studied for their possible use as passive transfer dosimeters for clinical proton beams. Evaluation of characteristics of these instruments, including the LET dependence of their response of proton energy, was carried out at the Institute of Theoretical and Experimental Physics. Results of absolute dose measurements were regarded as a preliminary step of dose intercomparison between ITEP and NIST. Measurements made in a number of experiments showed average agreement between the ITEP and NIST dosimetry standards to 2.5%.
