Denaturation Melting Research Articles

1829. The Paradox of KPC Bearing Strains of Klebsiella pneumoniae with the D179Y Substitution: Resistance to Ceftazidine/Avibactam (CZA) and Susceptibility to Meropenem (MEM)

BackgroundResistance to CZA is a serious limitation of treatment for KPC bearing Enterobacteriaceae infections. Recently, a single amino acid substitution (D179Y) was described in KPC-2 and KPC-3 bearing CZA-resistant K. pneumoniae recovered from patients failing treatment. In class A β-lactamases the D179 residue is located at the neck of the omega loop and is critical for KPC catalytic activity. In attempts to understand the evolution of substrate specificity in KPC-2, the D179Y variant of KPC-2 was shown to be resistant to CZA (ceftazidime forms a long-lived acyl enzyme with in KPC-2), but susceptible to MEM. A similar observation was made in clinical and laboratory-generated K. pneumoniae and E. coli strains bearing D179Y KPC-3. We were compelled to explore the catalytic mechanisms of susceptibility to MEM of the D179Y variants in KPC-2 vs. KPC-3.MethodsKPC-2, KPC-3, and D179Y in the respective KPC were cloned into an expression vector and the β-lactamase proteins were purified. 5 mg of each β-lactamase with and without MEM (1:1 molar ratio) was incubated for the time indicated and analyzed using the Quadrupole Time-of-Flight (QTOF) timed mass spectrometry for the reaction intermediates. To assess thermal stability, denaturation melting curves were run for 2 hours using 12 µM β-lactamase.ResultsThe D179Y variant forms prolonged acyl-complexes with meropenem in KPC-3 and KPC-2, which can be detected up to 24 hours (Figure 1). This prolonged trapping of meropenem by D179Y variants is not evident with the respective KPCs. Further, the tyrosine substitution at the D179 position (Tm = 48–52°C) destabilizes the KPC β-lactamases (TmKPC-2/3 = 52–56°C).ConclusionThese data suggest that MEM acts as a covalent β-lactamase inhibitor more than as a substrate for KPC-2 and -3. The mechanistic basis of paradoxical susceptibility to carbapenems provides an impetus to develop better therapeutic approaches to the increasing threat of carbapenem resistance and highlights how the rational design of novel β-lactam/β-lactamase inhibitors must consider mechanistic bases of resistance. Disclosures All Authors: No reported Disclosures.

ChemInform Abstract: Syntheses and Binding Studies of Oligonucleotides Containing N-Hydroxycarbamate Linkages: Potential DNA Cleaving Antisense Oligomers.

Abstract An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

Molecular Dynamics Simulations of the Denaturation and Refolding of an RNA Tetraloop

Tetraloops are very abundant structural elements of RNA that are formed by four nucleotides in a hairpin loop which is closed by a double stranded helical stem with some Watson-Crick base pairs. A tetraloop r(GCGAAGGC) was identified from the crystal structure of the central domain of 16S rRNA (727–730) in the Thermus thermophilus 30S ribosomal complex (1). The crystal structure of the 30S complex includes a total of 104 nucleotides from the central domain of the 16S rRNA and three ribosomal proteins S6, S15 and S18. Independent biochemical experiments have demonstrated that protein S15 plays the role in initiating the formation of the central domain of this complex. In the crystal, the tetraloop interacts with the protein S15 at two sites: one of them is associated with hydrogen bond interactions between residue His50 and nucleotide G730, and the other is associated with the occurrence of residue Arg53 beside A728. This paper uses molecular dynamics (MD) simulations to investigate the protein-dependent structural stability of the tetraloop and demonstrates the folding pathway of this tetraloop via melting denaturation and its subsequent refolding. Three important results are derived from these simulations: (i) The stability of nucleotide A728 appears to be protein dependent. Without the interaction with S15, A728 flips away from stacking with A729. (ii) The melting temperature demonstrated by the simulations is analogous to the results of thermodynamic experiments. In addition, the simulated folding of the tetraloop is stepwise: the native shape of the backbone is formed first; this is then followed by the formation of the Watson-Crick base pairs in the stem; and finally the hydrogen bonds and base stacking in the loop are formed. (iii) The tetraloop structure is similar to the crystal structure at salt concentrations of 0.1 M and 1.0 M used for the simulations, but the refolded structure at 0.1 M salt is more comparable to the crystal structure than at 1.0 M. The results from the simulations using both the Generalized Born continuum model and explicit solvent model (Particle Mesh Ewald) generate a similar pathway for unfolding/refolding of the tetraloop.

Syntheses and binding studies of oligonucleotides containing N-hydroxycarbamate linkages: potential DNA cleaving antisense oligomers

An efficient synthesis of N-hydroxycarbamate containing thymidine dimer 9 was accomplished and it was incorporated into automatic DNA syntheses to make thymidine 16mers T*-1, T*-2 and T*-3. Binding abilities of T*-1, T*-2 and T*-3 with poly (dA)-16mer were assayed by melting denaturation (Tm) studies of the corresponding duplexes. Iron binding ability of a thymidine oligomer with N-hydroxycarbamate linkages was studied by MALDI-MS. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides.

DNA Binding Properties of Some Cationic Acridine Derivatives

Four cationic acridine derivatives have been synthesized. The positively charged amine residue in one of these is connected directly on to the acridine nucleus and in three other acridines, the amines are connected via a 9-CH2 unit to acridine. We have investigated the binding of these acridines with mammalian DNA by absorption titration, UV- and induced-CD spectroscopy and competitive ethidium bromide displacement fluorescence assay. The effects on the DNA duplex denaturation melting temperatures upon binding of each one of these are also examined. The results obtained herein clearly show that the introduction of a -CH2 group in the im mediate vicinity of the interrelation moiety introduces alterations in the DNA binding characteristics of the resulting acridines.

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.