Computational Examination on the Active Site Structure of a (Peroxo)diiron(III) Intermediate in the Amine Oxygenase AurF.

In this work, we report the first computational investigation on the structure and properties of the (peroxo)diiron(III) intermediate of the AurF enzyme. Our calculations predict that, in the oxidized state of the AurF enzyme, the peroxo ligand is depicted in a µ-1,1-coordination mode with a protonated bridging ligand and is not in a µ-η(2):η(2) or µ-1,2 mode. Computed spectral data for the µ-1,1-coordination mode correlate well with experimental observations and unravel the potential of the energetics-spectroscopic approach adapted here.

C-H bond activation by metal-superoxo species: what drives high reactivity?

Metal-superoxo species are ubiquitous in metalloenzymes and bioinorganic chemistry and are known for their high reactivity and their ability to activate inert C-H bonds. The comparative oxidative abilities of M-O2(.-) species (M = Cr(III), Mn(III), Fe(III), and Cu(II)) towards C-H bond activation reaction are presented. These superoxo species generated by oxygen activation are found to be aggressive oxidants compared to their high-valent metal-oxo counterparts generated by O⋅⋅⋅O bond cleavage. Our calculations illustrate the superior oxidative abilities of Fe(III)- and Mn(III)-superoxo species compared to the others and suggest that the reactivity may be correlated to the magnetic exchange parameter.

On the controversy of metal ion composition on amine oxygenase (AurF): a computational investigation.

An energetics-spectroscopic approach based on DFT method reveals that the active site structure of AurF has {Fe(III)(2)O} central core with one protonated terminal glutamate.

Insights into the Dual Shuttle Catalytic Mechanism of Guanine Deaminase.

Guanine deaminases (GD) are essential enzymes that help in regulating the nucleobase pool. Since the deamination reaction can result in the accumulation of mutagenic bases that can lead to genomic instability, these enzymes are tightly regulated and are nonpromiscuous. Here, we delineate the basis of their substrate fidelity via entailing the reaction mechanism of deamination by employing density functional theory (DFT) calculations on NE0047, a GD from Nitrosomonas europaea. The results show that, unlike pyrimidine deaminases, which require a single glutamic acid as a proton shuttle, GDs involve two amino acids, E79 and E143 (numbering in NE0047), which control its reactivity. The hybrid quantum mechanics/molecular mechanics (QM/MM) calculations have shown that the first Zn-bound proton transfer to the N3 atom of the substrate is mediated by the E79 residue, and the second proton is transferred to the amine nitrogen of substrate via E143. Moreover, cluster models reveal that the crystallographic water molecules near the active site control the reactivity. A comparison with human GD reveals that the proposed catalytic mechanism is generic, and the knowledge generated here can be effectively applied to design selective inhibitors.

Structure-activity relationships of a caged thrombin binding DNA aptamer: insight gained from molecular dynamics simulation studies.

15-mer ssDNA aptamers play a vital role in the inhibition of alpha-thrombin in the blood clotting mechanism. It is of high importance to explore the structural factors controlling the inhibitory nature of the aptamer. Here we investigated the structure-function relationship of the anti-thrombin aptamer, as well as its 'caged' variant (2-(2-nitrophenyl)-propyl group (NPP)) by molecular dynamics simulations. The stability of the unmodified aptamer at different temperatures is examined in 2ns all-atom simulations and compared to experiment. The change in structure when introducing the photo-labile caged compound is analyzed, and the regiospecificity of this modification explained on atomic level. Removal of the photo-labile group leads to the reformation of the active aptamer structure from its inactive state. The mechanism for this formation process is a concerted movement of the aptamer backbone and some highly important bases. The binding of the aptamer to thrombin with regard to the 'caged' group is studied in an explicit simulation with the aptamer-thrombin complex and the reason for the binding/unbinding nature of the aptamer shown.

Synthesis, transacylation kinetics and computational chemistry of a set of arylacetic acid 1beta-O-acyl glucuronides.

Many widely-used non-steroidal anti-inflammatory agents (NSAIDs), e.g. ibuprofen, are extensively metabolised as their acyl glucuronides (AGs), and the reactivity of these AGs raises important questions regarding drug safety and toxicity. In order to understand better the structure-reactivity of these metabolites, we have performed a detailed study of the synthesis, structural analysis and computed transacylation reactivity of a set of acyl glucuronides (AGs) of phenylacetic acids with varying alpha-substitution. A selective acylation procedure was used to prepare all the desired 1-(phenyl)acetyl-beta-D-glucopyranuronic acids 9, 12, 13 and 15 as single 1beta-anomers in good yields. Their reactivity was measured using 1H NMR spectroscopy in pH 7.4 buffer: the dominance of transacylation over hydrolysis in this system was confirmed together with the measurement of half-lives of the 1beta-isomers of the AGs. The half-lives ranged from 20 min for compound 9 to 23 h for 15. The lack of any significant concentration dependence of the reactivity suggests that the main mechanism is intramolecular. A novel computational chemistry and modelling study was performed on both the ground states of the AGs and the transition states for acyl migration to search for correlations with the kinetic data and to probe the mechanistic detail of the acyl transfer. An excellent degree of correlation was found between the calculated activation energies and the rates of transacylation. Especially, transition state analysis provided for the first time a firm mechanistic explanation for the slower kinetics of the (S)-isomer AG 13 compared to the (R)-isomer 12, thus throwing important light on the pharmacokinetic behaviour of marketed NSAIDs.

QM/MM studies of Ni-Fe hydrogenases: the effect of enzyme environment on the structure and energies of the inactive and active states.

The catalytically active (Ni-SI and Ni-R) and inactive states (Ni-A and Ni-B) of Ni-Fe hydrogenases have been studied using density functional theory (DFT) methods. Both isolated clusters and clusters embedded in the enzyme have been used to model the Ni-A, Ni-B, Ni-SI and Ni-R states. The BP86 and B3LYP functionals were employed, and hybrid quantum mechanical (QM)/molecular mechanical (MM) methods were used for the embedded calculations. The QM/MM studies, rather than the isolated cluster calculations, were generally found to give structures which correlated better with X-ray data. The structure of the unready state (Ni-A), was correctly predicted by the QM/MM, but not by the isolated cluster calculation. Comparison with the observed crystal structure favoured the catalytically active state, Ni-SI, to be the protonated (Ni-SI(II)), rather than the unprotonated state (Ni-SI(I)). In the QM/MM studies, the binding of H(2) to Ni-SI(II) is preferred at the Ni (Ni-R(Ni)), rather than at the Fe centre (Ni-R(Fe)), in agreement with xenon binding studies, and in contrast to isolated cluster studies. These calculations cannot say with certainty which functional should be favoured, nor the preferred spin state of the catalytically active species. However, the lack of any predicted structure in which H(2) binds to the Fe centre, does favour a low spin state for Ni-SI(II), and the use of the BP86 functional. This is in agreement with recent high level ab initio calculations of a model of the Ni-SI(I) state.

High level ab initio and DFT calculations of models of the catalytically active Ni-Fe hydrogenases.

Multi-reference Møller-Plesset calculations of a model of the Ni-SI state of nickel-iron hydrogenase predict a singlet rather than a triplet state for this species, and show that it is better described with a BP86 rather than a B3LYP functional.

How are the ready and unready states of nickel-iron hydrogenase activated by H2? A density functional theory study.

We have explored possible mechanisms for the formation of the catalytically active Ni(a)-S state of the enzyme, nickel iron hydrogenase, from the Ni*(r) (ready) or Ni*(u) (unready) state, by reaction with H(2), using density functional theory calculations with the BP86 functional in conjunction with a DZVP basis set. We find that for the reaction of the ready state, which is taken to have an -OH bridge, the rate determining step is the cleavage of H(2) at the Ni(3+) centre with a barrier of approximately 15 kcal mol(-1). We take the unready state to have a -OOH bridge, and find that reaction with H(2) to form the Ni(r)-S state can proceed by two possible routes. One such path has a number of steps involving electron transfer, which is consistent with experiment, as is the calculated barrier of approximately 19 kcal mol(-1). The alternative pathway, with a lower barrier, may not be rate determining. Overall, our predictions give barriers in line with experiment, and allow details of the mechanism to be explored which are inaccessible from experiment.