Theoretical Study of the Arachidonic Acid Conversion into Leukotriene A4 Catalyzed by Human 5-Lipoxygenase : Hydroperoxidation and Epoxidation Mechanisms and Arachidonic Acid Active Site Access

Altres ajuts: acords transformatius de la UABInflammation is at the base of many different diseases. Leukotrienes (LTs) are pro-inflammatory mediators derived from arachidonic acid (AA), which play significant roles in acute inflammation. Lipoxins are specialized pro-resolving mediators (SPMs), also formed from AA, that promote the resolution of acute inflammation. However, if resolution fails, chronic inflammatory processes might develop. The enzyme human-5-lipoxygenase (5-LOX) catalyzes the biosynthesis of leukotriene named LTA but also intervenes in the formation of the lipoxin LXA. These two biological functions have made the 5-LOX isoform a current target for pharmaceutical investigations in several inflammatory-based diseases searching for inhibitors that block the leukotriene reaction pathway but not lipoxin's formation. However, the development of those selective inhibitors has been hampered by the lack of a crystal structure of human 5-LOX. In this work, we have built a complete solvated model of the human-5-LOX: AA Michaelis complex using, as initial coordinates, the human 5-LOX structure from the AlphaFold protein structure database. We aim to analyze at the molecular level the overall catalytic mechanism of 5-LOX that first converts AA into 5(S)-HpETE through a hydroperoxidation reaction and, second, transforms this hydroperoxide into LTA following an epoxidation process. Methodologically, we have performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. The free energy profiles for AA entrance into the 5-LOX's binding cavity have been calculated by steered molecular dynamics. This detailed molecular information can explain human-5-LOX's in vitro activity (without the presence of the membrane-embedded 5-lipoxygenase-activating protein) and help to design selective inhibitors favoring inflammation resolution

Unraveling the molecular details of the complete mechanism that governs the synthesis of prostaglandin G2 catalyzed by cyclooxygenase-2

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage AgreementCyclooxygenase-2 (COX-2) is the key enzyme involved in the synthesis pathway of prostaglandin G₂ (PGG₂) by transformation of arachidonic acid (AA). Although COX-₂ is one of the principal pharmacological targets by the implication of PGG₂2 in several human diseases, the classical all-radical mechanism proposed for COX-₂ catalysis has never been validated at the molecular level. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations were combined to analyze the six steps of the all-radical mechanism. The results show that O₂ addition on C₁₁ of AA can follow an antarafacial or suprafacial approach with respect to tyrosine 385, but only the antarafacial addition leads to the product with the correct 11R stereochemistry as established in the mechanistic proposal. Moreover, only the reaction pathway coming from the antarafacial intermediate describes a viable 8,12-cyclization to form the prostaglandin-like bicyclo endoperoxide that finally leads, by kinetic control, to PGG₂ with the 15S stereochemistry found experimentally. The formation of the more stable trans ring isomer of natural PGG₂ in an enzymatic environment is also explained. Our molecular analysis shows how COX-2 uses its relatively narrow channel in the active site to restrain certain conformational changes of AA and of the reaction intermediates, so that the PGG2 enzymatic synthesis turns out to be highly regiospecific and stereospecific. A more recent 10-step carbocation-based mechanistic proposal has been discarded

Comparing hydrolysis and transglycosylation reactions catalyzed by Thermus thermophilus β-glycosidase. A combined MD and QM/MM study

The synthesis of oligosaccharides and other carbohydrate derivatives is of relevance for the advancement of glycosciences both at the fundamental and applied level. For many years, glycosyl hydrolases (GHs) have been explored to catalyze the synthesis of glycosidic bonds. In particular, retaining GHs can catalyze a transglycosylation (T) reaction that competes with hydrolysis (H). This has been done either employing controlled conditions in wild type GHs or by engineering new mutants. The goal, which is to increase the T/H ratio, has been achieved with moderate success in several cases despite the fact that the molecular basis for T/H modulation are unclear. Here we have used QM(DFT)/MM calculations to compare the glycosylation, hydrolysis and transglycosylation steps catalyzed by wild type Thermus thermophilus β-glycosidase (family GH1), a retaining glycosyl hydrolase for which a transglycosylation yield of 36% has been determined experimentally. The three transition states have a strong oxocarbenium character and ring conformations between H and E. The atomic charges at the transition states for hydrolysis and transglycosylation are very similar, except for the more negative charge of the oxygen atom of water when compared to that of the acceptor Glc. The glycosylation transition state has a stronger S character than the deglycosylation ones and the proton transfer is less advanced. At the QM(PBE0/TZVP)/MM level, the TS for transglycosylation has shorter O4GLC-C1FUC (forming bond) distance and longer OE2 -C1 (breaking) distance than the hydrolysis one, although the HACC proton is closer to the Glu164 base in the hydrolysis TS. The QM(SCC-DFTB)/MM free energy maxima show the inverted situation, although the hydrolysis TS presents significant structural fluctuations. The 3-OHGLC group of the acceptor Glc (transglycosylation) and WAT432 (neighbor water in hydrolysis) are identified to stabilize the oxocarbenium transition states through interaction with O5 and O4 . The analysis of interaction suggests that perturbing the Glu392-Fuc interaction could increase the T/H ratio, either by direct mutation of this residue or indirectly as reported experimentally in the Asn390I and Phe401S cases. The molecular understanding of similarities and differences between hydrolysis and transglycosylation steps may be of help in the design of new biocatalysts for glycan synthesi

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

The synthesis of oligosaccharides and other carbohydrate derivatives is of relevance for the advancement of glycosciences both at the fundamental and applied level. For many years, glycosyl hydrolases (GHs) have been explored to catalyze the synthesis of glycosidic bonds. In particular, retaining GHs can catalyze a transglycosylation (T) reaction that competes with hydrolysis (H). This has been done either employing controlled conditions in wild type GHs or by engineering new mutants. The goal, which is to increase the T/H ratio, has been achieved with moderate success in several cases despite the fact that the molecular basis for T/H modulation are unclear. Here we have used QM(DFT)/MM calculations to compare the glycosylation, hydrolysis and transglycosylation steps catalyzed by wild type Thermus thermophilus β-glycosidase (family GH1), a retaining glycosyl hydrolase for which a transglycosylation yield of 36% has been determined experimentally. The three transition states have a strong oxocarbenium character and ring conformations between 4H3 and 4E. The atomic charges at the transition states for hydrolysis and transglycosylation are very similar, except for the more negative charge of the oxygen atom of water when compared to that of the acceptor Glc. The glycosylation transition state has a stronger SN2 character than the deglycosylation ones and the proton transfer is less advanced. At the QM(PBE0/TZVP)/MM level, the TS for transglycosylation has shorter O4GLC-C1FUC (forming bond) distance and longer OE2GLU338-C1FUC (breaking) distance than the hydrolysis one, although the HACC proton is closer to the Glu164 base in the hydrolysis TS. The QM(SCC-DFTB)/MM free energy maxima show the inverted situation, although the hydrolysis TS presents significant structural fluctuations. The 3-OHGLC group of the acceptor Glc (transglycosylation) and WAT432 (neighbor water in hydrolysis) are identified to stabilize the oxocarbenium transition states through interaction with O5FUC and O4FUC. The analysis of interaction suggests that perturbing the Glu392-Fuc interaction could increase the T/H ratio, either by direct mutation of this residue or indirectly as reported experimentally in the Asn390I and Phe401S cases. The molecular understanding of similarities and differences between hydrolysis and transglycosylation steps may be of help in the design of new biocatalysts for glycan synthesis

Accounting for the instantaneous disorder in the enzyme-substrate Michaelis complex to calculate the Gibbs free energy barrier of an enzyme reaction

Many enzyme reactions present instantaneous disorder. These dynamic fluctuations in the enzyme-substrate Michaelis complexes generate a wide range of energy barriers that cannot be experimentally observed, but that determine the measured kinetics of the reaction. These individual energy barriers can be calculated using QM/MM methods, but then the problem is how to deal with this dispersion of energy barriers to provide kinetic information. So far, the most usual procedure has implied the so-called exponential average of the energy barriers. In this paper, we discuss the foundations of this method, and we use the free energy perturbation theory to derive an alternative equation to get the Gibbs free energy barrier of the enzyme reaction. In addition, we propose a practical way to implement it. We have chosen four enzyme reactions as examples. In particular, we have studied the hydrolysis of a glycosidic bond catalyzed by the enzymeThermus thermophilusβ-glycosidase, and the mutant Y284P Ttb-gly, and the hydrogen abstraction reactions from C 13and C 7of arachidonic acid catalyzed by the enzyme rabbit 15-lipoxygenase-1

Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase : consequences for the positional specificity of oxygenation

In the present work we have combined homology modeling, protein-ligand dockings, quantum mechanics/molecular mechanics calculations and molecular dynamics simulations to generate human 5-lipoxygenase (5-LOX):arachidonic acid (AA) complexes consistent with the 5-lipoxygenating activity (which implies hydrogen abstraction at the C position). Our results suggest that both the holo and the apo forms of human Stable 5-LOX could accommodate AA in a productive form for 5-lipoxygenation. The former, in a tail-first orientation, with the AA carboxylate end interacting with Lys409, gives the desired structures with C close to the Fe-OH cofactor and suitable barrier heights for H abstraction. Only when using the apo form structure, a head-first orientation with the AA carboxylate close to His600 (a residue recently proposed as essential for AA positioning) is obtained in the docking calculations. However, the calculated barrier heights for this head-first orientation are in principle consistent with 5-LOX specificity, but also with 12/8 regioselectivity. Finally, long MD simulations give support to the recent hypothesis that the Phe177 + Tyr181 pair needs to close the active site access during the chemical reaction, and suggest that in the case of a head-first orientation Phe177 may be the residue interacting with the AA carboxylate

Functional Characterization of Mouse and Human Arachidonic Acid Lipoxygenase 15B (ALOX15B) Orthologs and of Their Mutants Exhibiting Humanized and Murinized Reaction Specificities

The arachidonic acid lipoxygenase 15B (ALOX15B) orthologs of men and mice form different reaction products when arachidonic acid is used as the substrate. Tyr603Asp+His604Val double mutation in mouse arachidonic acid lipoxygenase 15b humanized the product pattern and an inverse mutagenesis strategy murinized the specificity of the human enzyme. As the mechanistic basis for these functional differences, an inverse substrate binding at the active site of the enzymes has been suggested, but experimental proof for this hypothesis is still pending. Here we expressed wildtype mouse and human arachidonic acid lipoxygenase 15B orthologs as well as their humanized and murinized double mutants as recombinant proteins and analyzed the product patterns of these enzymes with different polyenoic fatty acids. In addition, in silico substrate docking studies and molecular dynamics simulation were performed to explore the mechanistic basis for the distinct reaction specificities of the different enzyme variants. Wildtype human arachidonic acid lipoxygenase 15B converted arachidonic acid and eicosapentaenoic acid to their 15-hydroperoxy derivatives but the Asp602Tyr+Val603His exchange murinized the product pattern. The inverse mutagenesis strategy in mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) humanized the product pattern with these substrates, but the situation was different with docosahexaenoic acid. Here, Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b also humanized the specificity but the inverse mutagenesis (Asp602Tyr+Val603His) did not murinize the human enzyme. With linoleic acid Tyr603Asp+His604Val substitution in mouse arachidonic acid lipoxygenase 15b humanized the product pattern but the inverse mutagenesis in human arachidonic acid lipoxygenase 15B induced racemic product formation. Amino acid exchanges at critical positions of human and mouse arachidonic acid lipoxygenase 15B orthologs humanized/murinized the product pattern with C20 fatty acids, but this was not the case with fatty acid substrates of different chain lengths. Asp602Tyr+Val603His exchange murinized the product pattern of human arachidonic acid lipoxygenase 15B with arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. An inverse mutagenesis strategy on mouse arachidonic acid lipoxygenase 15b (Tyr603Asp+His604Val exchange) did humanize the reaction products with arachidonic acid and eicosapentaenoic acid, but not with docosahexaenoic acid

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

Molecular Insights into the Regulation of 3-Phosphoinositide-Dependent Protein Kinase 1 : Modeling the Interaction between the Kinase and the Pleckstrin Homology Domains

The 3-phosphoinositide-dependent protein kinase 1 (PDK1) K465E mutant kinase can still activate protein kinase B (PKB) at the membrane in a phosphatidylinositol-3,4,5-trisphosphate (PIP3, PtdIns(3,4,5)P3) independent manner. To understand this new PDK1 regulatory mechanism, docking and molecular dynamics calculations were performed for the first time to simulate the wild-type kinase domain-pleckstrin homology (PH) domain complex with PH-in and PH-out conformations. These simulations were then compared to the PH-in model of the KD-PH(mutant K465E) PDK1 complex. Additionally, three KD-PH complexes were simulated, including a substrate analogue bound to a hydrophobic pocket (denominated the PIF-pocket) substrate-docking site. We find that only the PH-out conformation, with the PH domain well-oriented to interact with the cellular membrane, is active for wild-type PDK1. In contrast, the active conformation of the PDK1 K465E mutant is PH-in, being ATP-stable at the active site while the PIF-pocket is more accessible to the peptide substrate. We corroborate that both the docking-site binding and the catalytic activity are in fact enhanced in knock-in mouse samples expressing the PDK1 K465E protein, enabling the phosphorylation of PKB in the absence of PIP3binding

Synthetic Photoswitchable Neurotransmitters Based on Bridged Azobenzenes

Photoswitchable neurotransmitters of ionotropic kainate receptors were synthesized by tethering a glutamate moiety to disubstituted C2-bridged azobenzenes, which were prepared through a novel methodology that allows access to diazocines with higher yields and versatility. Because of the singular properties of these photochromes, photoisomerizable compounds were obtained with larger thermal stability for their inert cis isomer than for their biologically activity trans state. This enabled selective neuronal firing upon irradiation without background activity in the dark