Agnieszka Szarecka

Factors Affecting Conformation of (R,R)-Tartaric Acid Ester, Amide and Nitrile Derivatives. X-Ray Diffraction, Circular Dichroism, Nuclear Magnetic Resonance and Ab Initio Studies

Abstract Derivatives 2a–15a of (R,R)-tartaric acid (1a) with all combinations of methyl ester, amide, N-methylamide and N,N-dimethylamide groups, as well as the corresponding O,O′-dibenzoyl derivatives 1b–15b and nitriles 16–18 have been synthesized. Their conformations have been studied by the NMR and CD methods in solution as well as by X-ray diffraction in the crystalline state. The preference for planar. T conformation of the four carbon chain is observed under conditions restricting the α-hydroxyacid, ester or amide group to be nearly planar, this conformation being stabilized by intramolecular hydrogen bonds of the S(5) motif and the electrostatic CO/C(β)H and CN/C(β)H coplanar bond interactions. The C=O/C(α)-O bond system tends to be either synplanar (ester, acid), or antiplanar (ester, primary and secondary amide). Ab initio calculations allowed to demonstrate that for the isolated molecules of diamides 10a and 15a there is strong preference for gauche G+(a,a) conformers, the driving force being the formation of the hydrogen bonded six-membered cycles of the S(6) motif joining the OH and C=O groups from two different halves of the molecule. The results compare favourably with the experimental values derived from NMR spectra of 15a in nonpolar solvent. In the absence of intramolecular hydrogen bonding the N,N-dimethylamide group is better accomodated in a gauche G− conformer. This releases the nonbonded interaction due to the amide methyl group anti to the carbonyl group.

Dynamics of heteropentameric nicotinic acetylcholine receptor: implications of the gating mechanism.

The dynamics characteristics of the currently available structure of Torpedo nicotinic acetylcholine receptor (nAChR), including the extracellular, transmembrane, and intracellular domains (ICDs), were analyzed using the Gaussian Network Model (GNM) and Anisotropic Network Model (ANM). We found that a symmetric quaternary twist motion, reported previously in the literature in a homopentameric receptor (Cheng et al. J Mol Biol 2006;355:310–324; Taly et al. Biophys J 2005;88:3954–3965), occurred also in the heteropentameric Torpedo nAChR. We believe, however, that the symmetric twist alone is not sufficient to explain a large body of experimental data indicating asymmetry and subunit nonequivalence during gating. Here we report our results supporting the hypothesis that a combination of symmetric and asymmetric motions opens the gate. We show that the asymmetric motion involves tilting of the TM2 helices. Furthermore, our study reveals three additional aspects of channel dynamics: (1) loop A serves as an allosteric mediator between the ligand binding loops and those at the domain interface, particularly the linker between TM2 and TM3; (2) the ICD can modulate the pore dynamics and thus should not be neglected in gating studies; and (3) the F loops, which are peculiarly longer and poorly‐conserved in non‐α‐subunits, have important dynamical implications. Proteins 2007.

The Class D β-lactamase family: residues governing the maintenance and diversity of function

Class D β-lactamases, a major source of bacterial resistance to β-lactam antibiotic therapies, represent a distinct subset of the β-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ∼80 Class D β-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from α-, β- and γ-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of β-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of α-, β- and γ-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.

Absolute entropy and free energy of fluids using the hypothetical scanning method. I. Calculation of transition probabilities from local grand canonical partition functions

The hypothetical scanning (HS) method provides the absolute entropy and free energy from a Boltzmann sample generated by Monte Carlo, molecular dynamics or any other exact simulation procedure. Thus far HS has been applied successfully to magnetic and polymer chain models; in this paper and the following one it is extended to fluid systems by treating a Lennard-Jones model of argon. With HS a probability Pi approximating the Boltzmann probability of system configuration i is calculated with a stepwise reconstruction procedure, based on adding atoms gradually layer-by-layer to an initially empty volume, where they are replaced in their positions at i. At each step a transition probability (TP) is obtained from local grand canonical partition functions calculated over a limited space of the still unvisited (future) volume, the larger this space the better the approximation. Pi is the product of the step TPs, where ln Pi is an upper bound of the absolute entropy, which leads to upper and lower bounds for the...

Theoretical thermochemistry of the C60F18, C60F36, and C60F48 fluorofullerenes

Relative energies of C60FN fluorofullerenes are reproduced reasonably well at the B3LYP/6- 311G** level of theory employed in conjunction with isodesmic transfluorination reactions, although overestimation of steric repulsions among non-bonded atoms is evident for species with larger values of N. On the other hand, the MNDO method is found to be less suitable for studies of fluorofullerene thermochemistry. The gas-phase standard enthalpy of formation of the C60F18 species is predicted to lie between −1500 kJ mol−1 and −1400 kJ mol−1.

Structural Basis of Activity against Aztreonam and Extended Spectrum Cephalosporins for Two Carbapenem-Hydrolyzing Class D β-Lactamases from Acinetobacter baumannii

The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement.

Energetics, electronic structures and geometries of didehydroazines

When calibrated against the available experimental data for didehydrobenzenes, RB3LYP/cc-pVTZ, QCISD/cc-pVTZ, CCSD(T)/cc-pVTZ, and G3 electronic structure calculations provide reliable predictions of standard enthalpies and singlet—triplet splittings in all possible isomers of didehydroazines that (with a possible exception of 2,6-didehydropyridine) possess singlet ground states. Singlet didehydroazines with larger numbers of nitrogen atoms turn out to be more prone to ring opening, as indicated by the fact that out of the 6, 11, 6 and 3 possible didehydropyridines, didehydrodiazines, didehydrotriazines, and didehydrotetrazines, respectively, 5, 7, 2 and none are actually found. Immediate proximity of the nitrogen atom to the formally triple carbon—carbon bond confers decreased thermodynamic stabilities and smaller singlet—triplet splittings on the species of the 1,2-didehydro type. Some of the aza analogues of singlet 1,3-didehydrobenzene are as stable as their 1,2-didehydro counterparts. The only existing aza analogue of singlet 1,4-didehydrobenzene is 2,5-didehydropyrazine, which is particularly stable and possesses a large singlet—triplet splitting, making it a feasible synthetic target. The present calculations indicate that the experimental standard enthalpies of formation of pyrimidine and pyrazine are in error.

The structure of a doripenem‐bound OXA‐51 class D β‐lactamase variant with enhanced carbapenemase activity

OXA‐51 is a class D β‐lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii. Many variants of OXA‐51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high‐resolution structures of apo OXA‐51 and OXA‐51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl‐intermediate. The structure shows that acyl‐doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA‐24/40 (doripenem) and OXA‐23 (meropenem). In the OXA‐51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA‐51. Similarly, in the OXA‐51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA‐51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA‐51 and OXA‐51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA‐51 variants.

Gas-phase Conformational Analysis of (R,R)-Tartaric Acid, its Diamide, N,N,N′,N′- Tetramethyldiamide and Model Compounds

Abstract A review over most recent ab initio studies carried out at both RHF and MP2 levels on (R,R)-tartaric acid (TA), its diamide (DA), tetramethyldiamide (TMDA) and on three prototypic model systems (each of them constitutes a half of the respective parental molecule), i.e. 2-hydroxyacetic acid (HA), 2-hydroxyacetamide (HD) and 2-hydroxy-N,N-dimethylacetamide (HMD) is presented. (R,R)-tartaric acid and the derivatives have been completely optimized at RHF/6-31G * level and subsequently single-point energies of all conformers have been calculated with the use of second order perturbation theory according to the scheme: MP2/6-31G * //RHF/6-31G * . In the complete optimization of the model molecules at RHF level we have employed relatively large basis sets, augmented with polarisation and diffuse functions, namely 3-21G, 6-31G * , 6-31++G ** and 6-311++G ** . Electronic correlation has been included with the largest basis set used in this study, i.e. MP2/6-311++G ** //RHF/6-311++G ** single-point energy calculations have been performed. General confomational preferences of tartaric acid derivatives have been analysed as well as an attempt has been made to define main factors affecting the conformational behaviour of these molecules in the isolated state, in particular, the role and stability of intramolecular hydrogen bonding. In the case of the model compounds, our study principally concerned the conformational preferences and hydrogen bonding structure within the α-hydroxy-X moiety, where X=COOH, CONH 2 , CON(CH 3 ) 2 .

Clinical Variants of the Native Class D β-lactamase of Acinetobacter baumannii pose an emerging threat through increased hydrolytic activity against carbapenems

ABSTRACT The threat posed by the chromosomally encoded class D β-lactamase of Acinetobacter baumannii (OXA-51/66) has been unclear, in part because of its relatively low affinity and turnover rate for carbapenems. Several hundred clinical variants of OXA-51/66 have been reported, many with substitutions of active-site residues. We determined the kinetic properties of OXA-66 and five clinical variants with respect to a wide variety of β-lactam substrates. The five variants displayed enhanced activity against carbapenems and in some cases against penicillins, late-generation cephalosporins, and the monobactam aztreonam. Molecular dynamics simulations show that in OXA-66, P130 inhibits the side-chain rotation of I129 and thereby prevents doripenem binding because of steric clash. A single amino acid substitution at this position (P130Q) in the variant OXA-109 greatly enhances the mobility of both I129 and a key active-site tryptophan (W222), thereby facilitating carbapenem binding. This expansion of substrate specificity represents a very worrisome development for the efficacy of β-lactams against this troublesome pathogen.