The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.

The chemical transformations of formamide (NH(2)CHO), a molecule of prebiotic interest as a precursor for biomolecules, are investigated using methods of electronic structure computations and Rice-Rampserger-Kassel-Marcus (RRKM) theory. Specifically, quantum chemical calculations applying the coupled-cluster theory CCSD(T), whose energies are extrapolated to the complete basis set limit (CBS), are carried out to construct the [CH(3)NO] potential energy surface. RRKM theory is then used to systematically examine decomposition channels leading to the formation of small molecules including CO, NH(3), H(2)O, HCN, HNC, H(2), HNCO, and HOCN. The energy barriers for the decarboxylation, dehydrogenation, and dehydration processes are found to be in the range of 73-78 kcal/mol. H(2) loss is predicted to be a one-step process although a two-step process is competitive. CO elimination is found to prefer a two-step pathway involving the carbene isomer NH(2)CHO (aminohydroxymethylene) as an intermediate. This CO-elimination channel is also favored over the one-step H(2) loss, in agreement with experiment. The H(2)O loss is a multistep process passing through a formimic acid conformer, which subsequently undergoes a rate-limiting dehydration. The dehydration appears to be particularly favored in the low-temperature regime. The new feature identifies aminohydroxymethylene as a transient but crucial intermediate in the decarboxylation of formamide.

Ceftaroline, a recently approved β-lactam antibiotic for treatment of infections by methicillin-resistant Staphylococcus aureus (MRSA), is able to inhibit penicillin-binding protein 2a (PBP2a) by triggering an allosteric conformational change that leads to the opening of the active site. The opened active site is now vulnerable to inhibition by a second molecule of ceftaroline, an event that impairs cell-wall biosynthesis and leads to bacterial death. The triggering of the allosteric effect takes place by binding of the first antibiotic molecule 60 Å away from the active site of PBP2a within the core of the allosteric site. We document, by kinetic studies and by determination of three X-ray structures of the mutant variants of PBP2a that result in resistance to ceftaroline, that the effect of these clinical mutants is the disruption of the allosteric trigger in this important protein in MRSA. This is an unprecedented mechanism for antibiotic resistance.

Electron ionization of the DNA nucleobase, adenine, and the tRNA nucleobase, hypoxanthine, was investigated near the threshold region (∼5-20 eV) using a high-resolution hemispherical electron monochromator and a quadrupole mass spectrometer. Ion efficiency curves of the threshold regions and the corresponding appearance energies (AEs) are presented for the parent cations and the five most abundant fragment cations of each molecule. The experimental ionization energies (IEs) of adenine and hypoxanthine were determined to be 8.70 ± 0.3 eV and 8.88 ± 0.5 eV, respectively. Quantum chemical calculations (B3LYP/6-311+G(2d,p)) yielded a vertical IE of 8.08 eV and an adiabatic IE of 8.07 eV for adenine and a vertical IE of 8.51 eV and an adiabatic IE of 8.36 eV for hypoxanthine, and the lowest energy optimized structures of the fragment cations and their respective neutral species were calculated. The enthalpies of the possible reactions from the adenine and hypoxanthine cations were also obtained computationally, which assisted in determining the most likely electron ionization pathways leading to the major fragment cations. Our results suggest that the imidazole ring is more stable than the pyrimidine ring in several of the fragmentation reactions from both adenine and hypoxanthine. This electron ionization study contributes to the understanding of the biological effects of electrons on nucleobases and to the database of the electronic properties of biomolecules, which is necessary for modeling the damage of DNA in living cells that is induced by ionizing radiation.

Fourier transform-infrared spectroscopy (FT-IR) and temperature programmed desorption (TPD) have been used to examine the thermal processing of three isotopes of pure formamide ice (HCONH2, DCONH2, and HCOND2) adsorbed on a SiO2 interstellar grain analogue. Pure formamide ice on SiO2 nanoparticles displays at least three different phases that we interpret as a porous phase from ∼70-145 K, a compacted polycrystalline phase from ∼145-210 K, and a third slow diffusion and sublimation phase from ∼210-380 K. Possible dimerization is also discussed. Formamide desorption from the SiO2 grain surface is characterized by TPD of pure HCONH2 and mixed H2O:HCONH2 ices. Water desorbs at 160 K, and formamide has a TPD peak maximum at ∼228 K. A mean Eact of ∼14.7 kcal/mol (0.64 eV) was obtained using Redhead analysis, indicating strong intermolecular forces within formamide ice. The mixed H2O:HCONH2 ice TPD data suggests possible formamide accumulation if the grains are exposed to temperature cycles <180 K.