Understanding the Molecular Mechanism of the Ala-versus-Gly Concept Controlling the Product Specificity in Reactions Catalyzed by Lipoxygenases:A Combined Molecular Dynamics and QM/MM Study of Coral 8R-Lipoxygenase

Lipoxygenases (LOXs) are a family of enzymes that catalyze the highly specific hydroperoxidation of polyunsaturated fatty acids, such as arachidonic acid. Different stereo- or/and regioisomer hydroperoxidation products lead later to different metabolites that exert opposite physiological effects in the animal body and play a central role in inflammatory processes. The Gly-Ala switch of a single residue is crucial for the stereo- and regiocontrol in many lipoxygenases. Herein, we have combined molecular dynamics simulations with quantum mechanics/molecular mechanics calculations to study the hydrogen abstraction step and the molecular oxygen addition step of the hydroperoxidation reaction of arachidonic acid catalyzed by both wild-type Coral 8<i>R</i>-LOX and its Gly427Ala mutant. We have obtained a detailed molecular understanding of this Ala-versus-Gly concept. In wild type, molecular oxygen adds to C₈ of arachidonic acid with an <i>R</i> stereochemistry. In the mutant, Ala427 pushes Leu385, blocks the region over C₈, and opens an oxygen access channel now directed to C₁₂, where molecular oxygen is added with an <i>S</i> stereochemistry. Thus, the specificity turns out to be dramatically inverted. Since Leu385 is highly conserved among many lipoxygenase isoforms, this mechanism can be general, and we propose that the presence of such type of bulky and hydrophobic residues can be key in controlling the extreme regio- and stereospecificity of lipoxygenases and, as a consequence, their physiological effects

Depression, Somatization and Anxiety in Female Patients with Temporomandibular Disorders (TMD)

The aim of this research was to determine the possible differences in degrees of depression, somatization and anxiety between the acute and chronic female patients with temporomandibular disorders (TMD), and whether these differences exist in healthy female patients. Ninety female patients were involved in this research; 60 of them were TMD patients of the Dental Polyclinic, while other 30 females came for a rutine recall visit and had no problem related to TMD. Patients were aged 22 to 67 years, the average age being 38.5±12 years. All patients were asked to fill in the RDC/TMD protocol and three psychological tests (Emotions Profile Index, Somatization Scale and life Events Scale). Following the analysis of the RDC/TMD protocol and psychological tests, it was determined that the chronic female patients had higher depression and somatization scores in comparison with the acute patients (p<0.01); the acute patients self-perceive higher levels of anxiety in relation to the control group; furthermore, the patients reporting higher levels of depression were more inclined to somatization and had experienced a greater number of stress events in the past six months. It is beyond doubt that patients suffering from the TMD’s exhibit higher levels of depression, somatization and anxiety compared to the healthy ones, which proves that physiological factors may play a predisposing role in combination with reduced level of body tolerance to pain, and a decreased tolerance to stress

DFT analysis of rotational barriers, 1H and 13C NMR chemical shifts in neutral and protonated furfurylidenanilines

Rotational barriers were calculated for 4′-substituted neutral and protonated furfurylidenanilines (FA) by DFT methods and the corresponding equilibrium geometries were identified. Neutral FA have equilibrium dihedral angle values sensitive to the substituent. 1H and 13C Substituent Induced Shifts in FA were calculated from shielding tensors and compared with experimental ones. The experimental trends in 1H and 13C Substituent Induced Shifts of these compounds are well reproduced by the calculations. Differences in Mulliken charges for each position in neutral and protonated furfurylidenanilines were correlated with experimental 1H and 13C Substituent Induced Shifts. The experimental trends in 1H and 13C Substituent Induced Shifts were well reproduced by charge differences. Only the functional group 1H shifts do not correlate with the charges reflecting the variable effect of the anisotropy term due to the benzene ring. Protonation of FA removes this variability

Catalytic Mechanism of the Colistin Resistance Protein MCR-1

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyzes phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via trans-fer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involment of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases. </div