Exploring the catalytic cascade of cembranoid biosynthesis by combination of genetic engineering and molecular simulations.

While chemical steps involved in bioactive cembranoid biosynthesis have been examined, the corresponding enzymatic mechanisms leading to their formation remain elusive. In the tobacco plant, a putative cembratriene-ol synthase (CBTS) initiates the catalytic cascade that lead to the biosynthesis of cembratriene-4,6-diols, which displays antibacterial- and anti-proliferative activities. We report here on structural homology models, functional studies, and mechanistic explorations of this enzyme using a combination of biosynthetic and computational methods.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations.

Proton relay network in P450cam formed upon docking of putidaredoxin.

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O─O cleavage step remain partly elusive to date; effects observed in various enzyme mutants remain partly unexplained.

Actin stabilizing compounds show specific biological effects due to their binding mode.

Actin binding compounds are widely used tools in cell biology. We compare the biological and biochemical effects of miuraenamide A and jasplakinolide, a structurally related prototypic actin stabilizer. Though both compounds have similar effects on cytoskeletal morphology and proliferation, they affect migration and transcription in a distinctive manner, as shown by a transcriptome approach in endothelial cells.

Predicting the bioactive conformations of macrocycles: a molecular dynamics-based docking procedure with DynaDock.

Macrocyclic compounds are of growing interest as a new class of therapeutics, especially as inhibitors binding to protein-protein interfaces. As molecular modeling is a well-established complimentary tool in modern drug design, the number of attempts to develop reliable docking strategies and algorithms to accurately predict the binding mode of macrocycles is rising continuously. Standard molecular docking approaches need to be adapted to this application, as a comprehensive yet efficient sampling of all ring conformations of the macrocycle is necessary.

Ca2+ binding induced sequential allosteric activation of sortase A: An example for ion-triggered conformational selection.

The allosteric activation of the intrinsically disordered enzyme Staphylococcus aureus sortase A is initiated via binding of a Ca2+ ion. Although Ca2+ binding was shown to initiate structural changes inducing disorder-to-order transitions, the details of the allosteric activation mechanism remain elusive. We performed long-term molecular dynamics simulations of sortase A without (3 simulations of 1.

The low spin - high spin equilibrium in the S-state of the water oxidizing enzyme.

In Photosystem II (PSII), the MnCaO-cluster of the active site advances through five sequential oxidation states (S to S) before water is oxidized and O is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S and S is pH dependent, with a pK ≈ 8.

Activity of Topotecan toward the DNA/Topoisomerase I Complex: A Theoretical Rationalization.

Topotecan (TPT) is a nontoxic anticancer drug characterized by a pH-dependent lactone/carboxyl equilibrium. TPT acts on the covalently bonded DNA/topoisomerase I (DNA/TopoI) complex by intercalating between two DNA bases at the active site. This turns TopoI into a DNA-damaging agent and inhibits supercoil relaxation.

1,3-Dipolar Cycloaddition Reactions of Low-Valent Rhodium and Iridium Complexes with Arylnitrile N-Oxides.

The reactions between low-valent Rh(I) and Ir(I) metal-carbonyl complexes and arylnitrile oxides possess the electronic and structural features of 1,3-dipolar cycloadditions. Density functional theory (DFT) calculations on these reactions, involving both cyclopentadienyl and carboranyl ligands on the metal carbonyl, explain the ease of the chemical processes and the stabilities of the resulting metallaisoxazolin-5-ones. The metal-carbonyl bond has partial double bond character according to the Wiberg index calculated through NBO analysis, and so the reaction can be considered a normal 1,3-dipolar cycloaddition involving M═C bonds.

Natural-Product-Inspired Aminoepoxybenzoquinones Kill Members of the Gram-Negative Pathogen Salmonella by Attenuating Cellular Stress Response.

Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics.

Redox-coupled substrate water reorganization in the active site of Photosystem II-The role of calcium in substrate water delivery.

Photosystem II (PSII) catalyzes light-driven water splitting in nature and is the key enzyme for energy input into the biosphere. Important details of its mechanism are not well understood. In order to understand the mechanism of water splitting, we perform here large-scale density functional theory (DFT) calculations on the active site of PSII in different oxidation, spin and ligand states.

Why does Asn71 deamidate faster than Asn15 in the enzyme triosephosphate isomerase? Answers from microsecond molecular dynamics simulation and QM/MM free energy calculations.

Deamidation is the uncatalyzed process by which asparagine or glutamine can be transformed into aspartic acid or glutamic acid, respectively. In its active homodimeric form, mammalian triosephosphate isomerase (TPI) contains two deamidation sites per monomer. Experimental evidence shows that the primary deamidation site (Asn71-Gly72) deamidates faster than the secondary deamidation site (Asn15-Gly16).

Rationalization of the pKa values of alcohols and thiols using atomic charge descriptors and its application to the prediction of amino acid pKa's.

In a first step toward the development of an efficient and accurate protocol to estimate amino acids' pKa's in proteins, we present in this work how to reproduce the pKa's of alcohol and thiol based residues (namely tyrosine, serine, and cysteine) in aqueous solution from the knowledge of the experimental pKa's of phenols, alcohols, and thiols. Our protocol is based on the linear relationship between computed atomic charges of the anionic form of the molecules (being either phenolates, alkoxides, or thiolates) and their respective experimental pKa values. It is tested with different environment approaches (gas phase or continuum solvent-based approaches), with five distinct atomic charge models (Mulliken, Löwdin, NPA, Merz-Kollman, and CHelpG), and with nine different DFT functionals combined with 16 different basis sets.

Initiation of the reaction of deamidation in triosephosphate isomerase: investigations by means of molecular dynamics simulations.

Deamidation of asparagine is the spontaneous degradation of this residue into aspartic acid. The kinetics of this slow reaction is mainly dependent on the nature of the adjacent amino acid that follows asparagine in a peptide or protein primary sequence. In the homodimer triosephosphate isomerase (TPI), there are two main deamidation sites per subunit: Asn15-Gly16 and Asn71-Gly72 for which deamidation dynamics are known to be interrelated.

Cyclopolymerization reactions of diallyl monomers: exploring electronic and steric effects using DFT reactivity indices.

The regioselectivity in the cyclopolymerization of diallyl monomers is investigated using DFT-based reactivity indices. In the first part, the experimentally observed mode of cyclization (exo versus endo) of 11 selected radicals involved in this process is reproduced by the computation of activation energies, entropies, enthalpies, and Gibb's free energies for the 5- and 6-membered cyclization reactions. The application of a recently proposed energy partitioning of the activation barriers shows that the regioselectivity cannot be explained by the steric effect alone.