DszA Catalyzes C-S Bond Cleavage through N5-Hydroperoxyl Formation.

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N5OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

Engineering DszC Mutants from Transition State Macrodipole Considerations and Evolutionary Sequence Analysis.

We describe an approach to identify enzyme mutants with increased turnover using the enzyme DszC as a case study. Our approach is based on recalculating the barriers of alanine mutants through single-point energy calculations at the hybrid QM/MM level in the wild-type reactant and transition state geometries. We analyze the difference in the electron density between the reactant and transition state to identify sites/residues where electrostatic interactions stabilize the transition state over the reactants. We also assess the insertion of a unit probe charge to identify positions in which the introduction of charged residues lowers the barrier.

Deciphering the Catalytic Mechanism of Virginiamycin B Lyase with Multiscale Methods and Molecular Dynamics Simulations.

Due to the emergence of antibiotic resistance, the need to explore novel antibiotics and/or novel strategies to counter antibiotic resistance is of utmost importance. In this work, we explored the molecular and mechanistic details of the degradation of a streptogramin B antibiotic by virginiamycin B (Vgb) lyase of Staphylococcus aureus using classical molecular dynamics simulations and multiscale quantum mechanics/molecular mechanics methods. Our results were in line with available experimental kinetic information. Although we were able to identify a stepwise mechanism, in the wild-type enzyme, the intermediate is short-lived, showing a small barrier to decay to the product state. The impact of point mutations on the reaction was also assessed, showing not only the importance of active site residues to the reaction catalyzed by Vgb lyase but also of near positive and negative residues surrounding the active site. Using molecular dynamics simulations, we also predicted the most likely protonation state of the 3-hydroxypicolinic moiety of the antibiotic and the impact of mutants on antibiotic binding. All this information will expand our understanding of linearization reactions of cyclic antibiotics, which are crucial for the development of novel strategies that aim to tackle antibiotic resistance.

Unraveling the Reaction Mechanism of Russell's Viper Venom Factor X Activator: A Paradigm for the Reactivity of Zinc Metalloproteinases?

Snake venom metalloproteinases (SVMPs) are important drug targets against snakebite envenoming, the neglected tropical disease with the highest mortality worldwide. Here, we focus on Russell's viper (Daboia russelii), one of the "big four" snakes of the Indian subcontinent that, together, are responsible for ca. 50,000 fatalities annually. The "Russell's viper venom factor X activator" (RVV-X), a highly toxic metalloproteinase, activates the blood coagulation factor X (FX), leading to the prey's abnormal blood clotting and death. Given its tremendous public health impact, the WHO recognized an urgent need to develop efficient, heat-stable, and affordable-for-all small-molecule inhibitors, for which a deep understanding of the mechanisms of action of snake's principal toxins is fundamental. In this study, we determine the catalytic mechanism of RVV-X by using a density functional theory/molecular mechanics (DFT:MM) methodology to calculate its free energy profile. The results showed that the catalytic process takes place via two steps. The first step involves a nucleophilic attack by an in situ generated hydroxide ion on the substrate carbonyl, yielding an activation barrier of 17.7 kcal·mol-1, while the second step corresponds to protonation of the peptide nitrogen and peptide bond cleavage with an energy barrier of 23.1 kcal·mol-1. Our study shows a unique role played by Zn2+ in catalysis by lowering the pKa of the Zn2+-bound water molecule, enough to permit the swift formation of the hydroxide nucleophile through barrierless deprotonation by the formally much less basic Glu140. Without the Zn2+ cofactor, this step would be rate-limiting.

Animal Fatty Acid Synthase: A Chemical Nanofactory.

Fatty acids are crucial molecules for most living beings, very well spread and conserved across species. These molecules play a role in energy storage, cell membrane architecture, and cell signaling, the latter through their derivative metabolites. De novo synthesis of fatty acids is a complex chemical process that can be achieved either by a metabolic pathway built by a sequence of individual enzymes, such as in most bacteria, or by a single, large multi-enzyme, which incorporates all the chemical capabilities of the metabolic pathway, such as in animals and fungi, and in some bacteria. Here we focus on the multi-enzymes, specifically in the animal fatty acid synthase (FAS). We start by providing a historical overview of this vast field of research. We follow by describing the extraordinary architecture of animal FAS, a homodimeric multi-enzyme with seven different active sites per dimer, including a carrier protein that carries the intermediates from one active site to the next. We then delve into this multi-enzyme's detailed chemistry and critically discuss the current knowledge on the chemical mechanism of each of the steps necessary to synthesize a single fatty acid molecule with atomic detail. In line with this, we discuss the potential and achieved FAS applications in biotechnology, as biosynthetic machines, and compare them with their homologous polyketide synthases, which are also finding wide applications in the same field. Finally, we discuss some open questions on the architecture of FAS, such as their peculiar substrate-shuttling arm, and describe possible reasons for the emergence of large megasynthases during evolution, questions that have fascinated biochemists from long ago but are still far from answered and understood.

Modern Strategies for the Diversification of the Supply of Natural Compounds: The Case of Alkaloid Painkillers.

Plant-derived natural compounds have been used for treating diseases since prehistorical times. The supply of many plant-derived natural compounds for medicinal purposes, such as thebaine, morphine, and codeine, is primarily dependent on opium poppy crop harvesting. This dependency adds an extra risk factor to ensuring the supply chain because crops are highly susceptible to environmental conditions. Emerging technologies, such as biocatalysis, might help to solve this problem by diversifying the sources of supply of these compounds. Here we review the first committed step in the production of alkaloid painkillers, the production of S-norcoclaurine, and the enzymes involved. The improvement of these enzymes can be carried out experimentally by directed evolution and rational design strategies, supported by computational methods, to create variants that produce the S-norcoclaurine precursor for alkaloid painkillers in heterologous organisms, meeting the pharmaceutical industry standards and needs without depending on opium poppy crops.

Different Enzyme Conformations Induce Different Mechanistic Traits in HIV-1 Protease.

The influence of the dynamical flexibility of enzymes on reaction mechanisms is a cornerstone in biological sciences. In this study, we aim to 1) study the convergence of the activation free energy by using the first step of the reaction catalysed by HIV-1 protease as a case study, and 2) provide further evidence for a mechanistic divergence in this enzyme, as two different reaction pathways were seen to contribute to this step. We used quantum mechanics/molecular mechanics molecular dynamics simulations, on four different initial conformations that led to different barriers in a previous study. Despite the sampling, the four activation free energies still spanned a range of 5.0 kcal ⋅ mol-1 . Furthermore, the new simulations did confirm the occurrence of an unusual mechanistic divergence, with two different mechanistic pathways displaying equivalent barriers. An active-site water molecule is proposed to influence the mechanistic pathway.

Towards the Accurate Thermodynamic Characterization of Enzyme Reaction Mechanisms.

We employed QM/MM molecular dynamics (MD) simulations to characterize the rate-limiting step of the glycosylation reaction of pancreatic α-amylase with combined DFT/molecular dynamics methods (PBE/def2-SVP : AMBER). Upon careful choice of four starting active site conformations based on thorough reactivity criteria, Gibbs energy profiles were calculated with umbrella sampling simulations within a statistical convergence of 1-2 kcal ⋅ mol-1 . Nevertheless, Gibbs activation barriers and reaction energies still varied from 11.0 to 16.8 kcal ⋅ mol-1 and -6.3 to +3.8 kcal ⋅ mol-1 depending on the starting conformations, showing that despite significant state-of-the-art QM/MM MD sampling (0.5 ns/profile) the result still depends on the starting structure. The results supported the one step dissociative mechanism of Asp197 glycosylation preceded by an acid-base reaction by the Glu233, which are qualitatively similar to those from multi-PES QM/MM studies, and thus support the use of the latter to determine enzyme reaction mechanisms.

QM/MM Study of the Reaction Mechanism of Thermophilic Glucuronoyl Esterase for Biomass Treatment.

Hydrolysis of lignocellulosic biomass, composed of a lignin-carbohydrate-complex (LCC) matrix, is critical for producing bioethanol from glucose. However, current methods for LCC processing require costly and polluting processes. The fungal Thermothelomyces thermophila glucuronoyl esterase (TtGE) is a promising thermophilic enzyme that hydrolyses LCC ester bonds. This study describes the TtGE catalytic mechanism using QM/MM methods. Two nearly-degenerate rate-determining transition states were found, with barriers of 16 and 17 kcal ⋅ mol-1 , both with a zwitterionic nature that results from a proton interplay from His346 to either the Ser213-hydroxyl or the lignin leaving group and the rehybridisation of the ester moiety of the substrate to an alkoxide. An oxyanion hole, characteristic of esterases, was provided by the conserved Arg214 through its backbone and sidechain. Our work further suggests that a mutation of Glu267 to a non-negative residue will decrease the energetic barrier in ca. -5 kcal ⋅ mol-1 , improving the catalytic rate of TtGE.

Role of Enzyme and Active Site Conformational Dynamics in the Catalysis by α-Amylase Explored with QM/MM Molecular Dynamics.

We assessed enzyme:substrate conformational dynamics and the rate-limiting glycosylation step of a human pancreatic α-amylase:maltopentose complex. Microsecond molecular dynamics simulations suggested that the distance of the catalytic Asp197 nucleophile to the anomeric carbon of the buried glucoside is responsible for most of the enzyme active site fluctuations and that both Asp197 and Asp300 interact the most with the buried glucoside unit. The buried glucoside binds either in a 4C1 chair or 2SO skew conformations, both of which can change to TS-like conformations characteristic of retaining glucosidases. Starting from four distinct enzyme:substrate complexes, umbrella sampling quantum mechanics/molecular mechanics simulations (converged within less than 1 kcal·mol-1 within a total simulation time of 1.6 ns) indicated that the reaction occurrs with a Gibbs barrier of 13.9 kcal·mol -1, in one asynchronous concerted step encompassing an acid-base reaction with Glu233 followed by a loose SN2-like nucleophilic substitution by the Asp197. The transition state is characterized by a 2H3 half-chair conformation of the buried glucoside that quickly changes to the E3 envelope conformation preceding the attack of the anomeric carbon by the Asp197 nucleophile. Thermodynamic analysis of the reaction supported that a water molecule tightly hydrogen bonded to the glycosidic oxygen of the substrate at the reactant state (∼1.6 Å) forms a short hydrogen bond with Glu233 at the transition state (∼1.7 Å) and lowers the Gibbs barrier in over 5 kcal·mol-1. The resulting Asp197-glycosyl was mostly found in the 4C1 conformation, although the more endergonic B3,O conformation was also observed. Altogether, the combination of short distances for the acid-base reaction with the Glu233 and for the nucleophilic attack by the Asp197 nucleophile and the availability of water within hydrogen bonding distance of the glycosidic oxygen provides a reliable criteria to identify reactive conformations of α-amylase complexes.

Root-knot nematodes (Meloidogyne spp.) a threat to agriculture in Mexico: biology, current control strategies, and perspectives.

Root-knot nematodes (RKN) are sedentary parasites of the roots of plants and are considered some of the most damaging pests in agriculture. Since RKN target the root vascular system, they provoke host nutrient deprivation and defective water transport, causing above-ground symptoms of growth stunting, wilting, chlorosis, and reduced crop yields. In Mexico RKN infestations are primarily dealt with by treating with synthetic chemically based nematicides that are preferred by farmers over available bioproducts. However, due to environmental and human health concerns chemical control is increasingly restricted. Biological control of RKNs can help reduce the use of chemical nematicides as it is achieved with antagonistic organisms, mainly bacteria, fungi, other nematodes, or consortia of diverse microorganisms, which control nematodes directly by predation and parasitism at different stages: eggs, juveniles, or adults; or indirectly by the action of toxic diffusible inhibitory metabolites. The need to increase agricultural production and reduce negative environmental impact creates an opportunity for optimizing biological control agents to suppress nematode populations, but this endeavour remains challenging as researchers around the world try to understand diverse control mechanisms, nematode and microbe life cycles, ecology, metabolite production, predatory behaviours, molecular and biochemical interactions, in order to generate attractive products with the approval of local regulatory bodies. Here, we provide a brief review of the biology of the genus Meloidogyne, biological control strategies, and a comparison between chemical and bioproducts in the Mexican market, and guidelines emitted by national agencies to ensure safety and effectiveness of new developments.

Necessity is the Mother of Invention: A Remote Molecular Bioinformatics Practical Course in the COVID-19 Era.

The COVID-19 pandemic has brought many challenges to human beings, related to not only health and way of life but also teaching because of the interruption of the standard training at universities imposed by lockdowns. Concerning the latter, the academic community had to reinvent itself, in many ways, to carry on with prepandemic education. This article focuses on the use of modern technology and software to create a virtual, highly interactive classroom where a remote but still hands-on course on molecular bioinformatics can be taught, motivating the university students and helping them learn the course contents without significant compromises imposed by successive lockdowns. We implemented such a virtual hands-on molecular bioinformatics course in the second semester of the 2020/2021 academic year. Furthermore, we compared the learning outcomes with those for the earlier editions of the same course in the pre-COVID-19 era, in which the more traditional teaching method was used where all teaching was delivered with physically present lecturers. The virtual classroom proposed here allowed the students to develop skills close to, although slightly below, those obtained with physically present learning.

Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides.

The membrane-active nature of phospholipase A2-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA2 toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A2 isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Novel Pet-Degrading Enzymes: Structure-Function from a Computational Perspective.

The bacterium strain Ideonella sakaiensis 201-F6 is able to hydrolyze low-crystallinity PET films at 30 °C due to two enzymes named PETase and MHETase. Since its discovery, many efforts have been dedicated to elucidating the structure and features of those two enzymes, and various authors have highlighted the necessity to optimize both the substrate binding site and the global structure in order to enhance the stability and catalytic activity of these PET biocatalysts so as to make them more suitable for industrial applications. In this review, the strategies adopted by different research groups to investigate the structure and functionality of both PETase and MHETase in depth are described, emphasizing the advantages provided by the use of computational methods to complement and drive experiments. Subsequently, the modifications implemented with protein engineering are discussed. The versatility of the enzymes secreted by I. sakaiensis enables the prediction that they will find several applications in the disposal of PET debris, encouraging a prioritization of efforts in this prolific research field.

The Catalytic Mechanism of the Retaining Glycosyltransferase Mannosylglycerate Synthase.

To protect their intracellular proteins, extremophile microorganisms synthesize molecules called compatible solutes. These molecules are the result of the attachment of a small negatively charged molecule to a sugar molecule. It has been found that these molecules, not only protect the microorganism against osmotic stress but also against other extreme conditions. They can also confer protection against extreme conditions to isolated enzymes from different organisms making them an exciting prospect for potential biotechnological applications. One of the most widespread compatible solute in hyperthermophile organisms is the molecule 2-O-α-D-mannosyl-D-glycerate (MG). In addition to confer protection to proteins against extreme conditions, MG was found to prevent Alzheimer's ß-amyloid aggregation and reduce α-synuclein fibril formation in Parkinson's disease. In this work we studied, using computational methods, the catalytic mechanism of the synthesis of MG by the enzyme mannosylglycerate synthase (MGS) from the thermophilic bacteria Rhodothermus marinus.

Unraveling the cGAS catalytic mechanism upon DNA activation through molecular dynamics simulations.

Cyclic GMP-AMP Synthase (cGAS) is activated upon DNA binding and catalyzes the synthesis of 2',3'-cGAMP from GTP and ATP. This cyclic dinucleotide is a messenger that triggers the autoimmune system of eukaryotic cells. In this study, we propose a Molecular Dynamics (MD) investigation of cGAS activation. We notably provide insights into the motion of the activation loop, both from a mechanical point of view and considering its role in the catalysis of cGAMP production. We finally shed light on the reaction resulting in cGAMP synthesis. Two possible catalytic routes (referred to as GTP-ATP and ATP-GTP) are proposed based on the active site occupancy, paving the way toward further exploration of the reaction mechanism.

Combined in silico and in vitro studies to identify novel antidiabetic flavonoids targeting glycogen phosphorylase.

Novel pharmacological strategies for the treatment of diabetic patients are now focusing on inhibiting glycogenolysis steps. In this regard, glycogen phosphorylase (GP) is a validated target for the discovery of innovative antihyperglycemic molecules. Natural products, and in particular flavonoids, have been reported as potent inhibitors of GP at the cellular level. Herein, free-energy calculations and microscale thermophoresis approaches were performed to get an in-depth assessment of the binding affinities and elucidate intermolecular interactions of several flavonoids at the inhibitor site of GP. To our knowledge, this is the first study indicating genistein, 8-prenylgenistein, apigenin, 8-prenylapigenin, 8-prenylnaringenin, galangin and valoneic acid dilactone as natural molecules with high inhibitory potency toward GP. We identified: i) the residues Phe285, Tyr613, Glu382 and/or Arg770 as the most relevant for the binding of the best flavonoids to the inhibitor site of GP, and ii) the 5-OH, 7-OH, 8-prenyl substitutions in ring A and the 4'-OH insertion in ring B to favor flavonoid binding at this site. Our results are invaluable to plan further structural modifications through organic synthesis approaches and develop more effective pharmaceuticals for Type 2 Diabetes treatment, and serve as the starting point for the exploration of food products for therapeutic usage, as well as for the development of novel bio-functional food and dietary supplements/herbal medicines.

Chromeno[3,4-b]xanthones as First-in-Class AChE and Aß Aggregation Dual-Inhibitors.

Alzheimer's disease (AD) is a complex multifactorial disorder, mainly characterized by the progressive loss of memory and cognitive, motor, and functional capacity. The absence of effective therapies available for AD alongside the consecutive failures in the central nervous system (CNS) drug development has been motivating the search for new disease-modifying therapeutic strategies for this disease. To address this issue, the multitarget directed ligands (MTDLs) are emerging as a therapeutic alternative to target the multiple AD-related factors. Following this concept, herein we describe the design, synthesis, and biological evaluation of a family of chromeno[3,4-b]xanthones as well as their (E)-2-[2-(propargyloxy)styryl]chromone precursors, as first-in-class acetylcholinesterase (AChE) and ß-amyloid (Aß) aggregation dual-inhibitors. Compounds 4b and 10 emerged as well-balanced dual-target inhibitors, with IC50 values of 3.9 and 2.9 µM for AChE and inhibitory percentages of 70 and 66% for Aß aggregation, respectively. The molecular docking showed that most of the compounds bound to AChE through hydrogen bonds with residues of the catalytic triad and π-stacking interactions between the main scaffold and the aromatic residues present in the binding pocket. The interesting well-balanced activities of these compounds makes them interesting templates for the development of new multitarget compounds for AD.

Assessing the validity of DLPNO-CCSD(T) in the calculation of activation and reaction energies of ubiquitous enzymatic reactions.

The domain-based local pair natural orbital coupled-cluster with single, double, and perturbative triples excitation (DLPNO-CCSD(T)) method was employed to portray the activation and reaction energies of four ubiquitous enzymatic reactions, and its performance was confronted to CCSD(T)/complete basis set (CBS) to assess its accuracy and robustness in this specific field. The DLPNO-CCSD(T) results were also confronted to those of a set of density functionals (DFs) to understand the benefit of implementing this technique in enzymatic quantum mechanics/molecular mechanics (QM/MM) calculations as a second QM component, which is often treated with DF theory (DFT). On average, the DLPNO-CCSD(T)/aug-cc-pVTZ results were 0.51 kcal·mol-1 apart from the canonic CCSD(T)/CBS, without noticeable biases toward any of the reactions under study. All DFs fell short to the DLPNO-CCSD(T), both in terms of accuracy and robustness, which suggests that this method is advantageous to characterize enzymatic reactions and that its use in QM/MM calculations, either alone or in conjugation with DFT, in a two-region QM layer (DLPNO-CCSD(T):DFT), should enhance the quality and faithfulness of the results.

Structural Specificity of Flavonoids in the Inhibition of Human Fructose 1,6-Bisphosphatase.

Liver fructose 1,6-bisphosphatase (FBPase) is a recognized regulatory enzyme of the gluconeogenesis pathway, which has emerged as a valid target to control gluconeogenesis-mediated overproduction of glucose. As such, the management of diabetes with FBPase inhibitors represents a potential alternative for the currently used antidiabetic agents. In this study, the FBPase inhibition of a panel of 55 structurally related flavonoids was tested, through a microanalysis screening system. Then, a subset of seven active inhibitors and their close chemical relatives were further evaluated by molecular dynamics (MD) simulations using a linear interaction energy (LIE) approach. The results obtained showed that D14 (herbacetin) was the most potent inhibitor, suggesting that the presence of -OH groups at the C-3, C-4', C-5, C-7, and C-8 positions, as well as the double bond between C-2 and C-3 and the 4-oxo function at the pyrone ring, are favorable for the intended effect. Furthermore, D14 (herbacetin) is stabilized by a strong interaction with the Glu30 side chain and the Thr24 backbone of FBPase. This is the first investigation studying the in vitro inhibitory effect of a panel of flavonoids against human liver FBPase, thus representing a potentially important step for the search and design of novel inhibitors of this enzyme.