DFT and QM/MM computations of allylic C-H hydroxylation versus C=C epoxidation in cyclohexene and propene by Compound I of P450cam demonstrate that the relative barriers for the oxidative processes themselves are not good predictors of the observed selectivity. However, a kinetic expression previously developed (Kozuch, S.; Shaik, S. J. Am. Chem. Soc. 2006, 128, 3355) for catalytic cycles under steady-state conditions, predicts, in accord with experiment, that propene will undergo exclusive C=C epoxidation, while cyclohexene will undergo both reactions with a small preference for epoxidation. The model expression for the effective barrier of the cycle forms a general basis for understanding and predicting the selectivity of P450 isozymes.

Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron-oxo species, called Compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substratefree or containing one and two molecules of diazepam as a substrate. Mössbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe-S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH---S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe-S bond, and as such, the presence of the substrate leads to a shorter Fe-S bond and it prevents the localization of the radical on the sulfur. This diazepam-Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (called also cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues.

Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C-H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme.

Density functional theory calculations have been performed to elucidate the factors that influence the regioselectivity of toluene hydroxylation by a model of the active species of cytochrome P450 enzymes, so‐called Compound I (Cpd I). Cpd I can hydroxylate the benzylic C–H and generate benzyl alcohol, or it can activate the phenyl group and produce p‐cresol and p‐methylcyclohexanone products. The reactions take place via two‐state reactivity on competing doublet and quartet spin state surfaces. In the gas phase, the benzyl alcohol is preferred over p‐cresol by more than three orders of magnitude. Environmental perturbations, namely, NH···S hydrogen bonding to the thiolate ligand and bulk polarity of the protein, lower this preference to roughly 10:1 in favour of benzyl alcohol. Substitution of methyl hydrogen atoms by deuterium atoms raises the barriers that lead to benzyl alcohol without affecting those leading to p‐cresol. Therefore combining toluene deuteration with the effects of NH···S hydrogen bonding and bulk polarity lowers the free energy difference between the two processes to only 0.4 kcal mol–1, and the two processes become competitive. These results as well as the calculated kinetic isotope effects are in good general agreement with experimental data. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

The unchecked dispersal of antipersonnel landmines since the late 19th century has resulted in large areas contaminated with these explosive devices, creating a substantial worldwide humanitarian safety risk. The main obstacle to safe and effective landmine removal is the identification of their exact location, an activity that currently requires entry of personnel into the minefields; to date, there is no commercialized technology for an efficient stand-off detection of buried landmines. In this article, we describe the optimization of a microbial sensor strain, genetically engineered for the remote detection of 2,4,6-trinitrotoloune (TNT)-based mines. This bioreporter, designed to bioluminescence in response to minute concentrations of either TNT or 2,4-dinitotoluene (DNT), was immobilized in hydrogel beads and optimized for dispersion over the minefield. Following modifications of the hydrogel matrix in which the sensor bacteria are encapsulated, as well as their genetic reporting elements, these sensor bacteria sensitively detected buried 2,4-dinitrotoluene in laboratory experiments. Encapsulated in 1.5 mm 2% alginate beads containing 1% polyacrylic acid, they also detected the location of a real metallic antipersonnel landmine under field conditions. To the best of our knowledge, this is the first report demonstrating the detection of a buried landmine with a luminescent microbial bioreporter.

Elevated levels of activated protein kinase B (PKB/Akt) have been detected in many types of cancer. Substrate-based peptide inhibitors have the advantage of selectivity due to their extensive interactions with the kinase-specific substrate binding site but often lack necessary pharmacological properties. Chemical modifications of potent peptide inhibitors, such as cyclization, may overcome these drawbacks while maintaining potency. We present an extensive structure-activity relationship (SAR) study of a potent peptide-based PKB/Akt inhibitor. Two backbone cyclic (BC) peptide libraries with varying modes of cyclization, bridge chemistry, and ring size were synthesized and evaluated for in vitro PKB/Akt inhibition. Backbone-to-backbone urea BC peptides were more potent than N-terminus-to-backbone amide BC peptides. Several analogues were up to 10-fold more active than the parent linear peptide. Some activity trends could be rationalized using computational surface mapping of the PKB/Akt kinase catalytic domain. The novel molecules have enhanced pharmacological properties which make them promising lead candidates.

Catalytic nucleic acids consisting of a bis‐Zn2+‐pyridyl‐salen‐type ([di‐ZnII 3,5 bis(pyridinylimino) benzoic acid]) complex conjugated to the ATP aptamer act as ATPase‐mimicking catalysts (nucleoapzymes). Direct linking of the Zn2+ complex to the 3′‐ or 5′‐end of the aptamer (nucleoapzymes I and II) or its conjugation to the 3′‐ or 5′‐end of the aptamer through bis‐thymidine spacers (nucleoapzymes III and IV) provided a set of nucleoapzymes exhibiting variable catalytic activities. Whereas the separated bis‐Zn2+‐pyridyl‐salen‐type catalyst and the ATP aptamer do not show any noticeable catalytic activity, the 3′‐catalyst‐modified nucleoapzyme (nucleoapzyme IV) and, specifically, the nucleoapzyme consisting of the catalyst linked to the 3′‐position through the spacer (nucleoapzyme III) reveal enhanced catalytic features in relation to the analogous nucleoapzyme substituted at the 5′‐position (kcat=4.37 and 6.88 min−1, respectively). Evaluation of the binding properties of ATP to the different nucleoapzyme and complementary molecular dynamics simulations suggest that the distance separating the active site from the substrate linked to the aptamer binding site controls the catalytic activities of the different nucleoapzymes.

Summary Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post‐transcriptional level. Following transcription, the polycistronic pre‐RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N6‐methylation of adenosine ribonucleotides (m6A‐RNA) in plant mitochondria. m6A is a prevalent modification in nuclear‐encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m6A mediates structural switches that affect RNA stability and/or activity. Using m6A‐pulldown/RNA‐seq (m6A‐RIP‐seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m6A‐RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m6A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non‐coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m6A modifications, N6‐methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.