5 research outputs found
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<sup>III</sup>–OOH or HO–Fe<sup>V</sup>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 ironÂ(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<sup>–</sup> and Cl<sup>–</sup> form the most stable complex with <b>6</b>, while the linear N<sub>3</sub><sup>–</sup> interacts most favorably with <b>7</b>. The trigonal NO<sub>3</sub><sup>–</sup> and tetrahedral ClO<sub>4</sub><sup>–</sup> 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<sup>–</sup> 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<sub>BM3</sub> Variants
Cytochrome
P450<sub>BM3</sub> 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<sub>BM3</sub> 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<sub>BM3</sub> variants therefore expedites site-directed mutagenesis
studies aimed at developing thermostability