Alchemical Free-Energy Calculations of Watson–Crick and Hoogsteen Base Pairing Interconversion in DNA

Hoogsteen (HG) base pairs have a transient nature and can be structurally similar to Watson–Crick (WC) base pairs, making their occurrence and thermodynamic stability difficult to determine experimentally. Herein, we employed the restrain–free-energy perturbation–release (R-FEP-R) method to calculate the relative free energy of the WC and HG base pairing modes in isolated and bound DNA systems and predict the glycosyl torsion conformational preference of purine bases. Notably, this method does not require prior knowledge of the transition pathway between the two end states. Remarkably, relatively fast convergence was reached, with results in excellent agreement with experimental data for all the examined DNA systems. The R-REP-R method successfully determined the stability of HG base pairing and more generally, the conformational preference of purine bases, in these systems. Therefore, this computational approach can help to understand the dynamic equilibrium between the WC and HG base pairing modes in DNA

A DFT Study of the <i>cis</i>-Dihydroxylation of Nitroaromatic Compounds Catalyzed by Nitrobenzene Dioxygenase

The mechanism of <i>cis</i>-dihydroxylation of nitrobenzene and 2-nitrotoluene catalyzed by nitrobenzene 1,2-dioxygenase (NBDO), a member of the naphthalene family of Rieske non-heme iron dioxygenases, was studied by means of the density functional theory method using four models of the enzyme active site. Different possible reaction pathways for the substrate dioxygenation initiated either by the Fe^III–OOH or HO–Fe^VO attack on the aromatic ring were considered and the computed activation barriers compared with the Gibbs free energy of activation for the oxygen–oxygen cleavage leading to the formation of the iron­(V)–oxo species from its ferric hydroperoxo precursor. The mechanism of the substrate <i>cis</i>-dihydroxylation leading to the formation of a <i>cis</i>-dihydrodiol was then investigated, and the most feasible mechanism was found to be starting with the attack of the high-valent iron–oxo species on the substrate ring yielding a radical intermediate, which further evolves toward the final product

Molecular Dynamics Simulation of Nitrobenzene Dioxygenase Using AMBER Force Field

Molecular dynamics simulation of the oxygenase component of nitrobenzene dioxygenase (NBDO) system, a member of the naphthalene family of Rieske nonheme iron dioxygenases, has been carried out using the AMBER force field combined with a new set of parameters for the description of the mononuclear nonheme iron center and iron–sulfur Rieske cluster. Simulation results provide information on the structure and dynamics of nitrobenzene dioxygenase in an aqueous environment and shed light on specific interactions that occur in its catalytic center. The results suggest that the architecture of the active site is stabilized by key hydrogen bonds, and Asn258 positions the substrate for oxidation. Analysis of protein–water interactions reveal the presence of a network of solvent molecules at the entrance to the active site, which could be of potential catalytic importance

Anion Binding by Electron-Deficient Arenes Based on Complementary Geometry and Charge Distribution

Extended electron-deficient arenes are investigated as potential neutral receptors for polyanions. Anion binds via σ interaction with extended arenes, which are composed solely of C and N ring atoms and CN substituents. As a result, the positive charge on the aromatic C is enhanced, consequently maximizing binding strength. Selectivity is achieved because different charge distributions can be obtained for target anions of a particular geometry. The halides F^– and Cl^– form the most stable complex with <b>6</b>, while the linear N₃^– interacts most favorably with <b>7</b>. The trigonal NO₃^– and tetrahedral ClO₄^– fit the 3-fold rotational axis of <b>6</b> but do not form stable complexes with <b>5</b> and <b>7</b>. The Y-shaped HCOO^– forms complexes with <b>4</b>, <b>5</b>, and <b>7</b>, with the latter being the most stable. Thus, the anion complexes exhibit strong binding and the best geometrical fit between guest and host, reminiscent of Lego blocks

Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450_BM3 Variants

Cytochrome P450_BM3 is a heme-containing enzyme from <i>Bacillus megaterium</i> that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450_BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450_BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability