scholarly journals Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

2021 ◽  
Author(s):  
Keisuke Fujiyama ◽  
Naoki Kato ◽  
Suyong Re ◽  
Kiyomi Kinugasa ◽  
Kohei Watanabe ◽  
...  
Keyword(s):  
Natural Products ◽  
Crystal Structures ◽  
Molecular Mechanism ◽  
Density Functional ◽  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Bioactive Molecules ◽  
Docking Simulations ◽  
Primary And Secondary Metabolites ◽  
Binding Models

SummaryMolecular chirality, discovered by Louis Pasteur in the middle of the 19th century1, is found in most primary and secondary metabolites. Particularly, the so-called natural products are rich in chiral centres2. The stereochemistry of natural products is strictly recognized in living organisms, and is thus closely related to their biological functions. The Diels–Alder (DA) reaction, which forms a six-membered ring with up to four chiral centres, is a fundamental practical reaction for C–C bond formation in synthetic chemistry3. Nature has also adopted this reaction to elaborate the complex structures of natural products using enzymes derived from various progenitor proteins4-7. Although enzymes catalysing the DA reaction, Diels–Alderases (DAases), have attracted increasing attention, little is known about the molecular mechanism by which they control the stereochemistry and perform catalysis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyse intramolecular DA reactions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin8,9. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided dynamic binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, explaining the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyse the stereoselective DA reaction. This study highlights the distinct molecular mechanisms of the enzymatic DA reaction and its stereoselectivity experimentally and computationally. We anticipate that clarified molecular mechanism herein provides not only the basic understanding how these important enzymes work but also the guiding principle to create artificial enzymes that produce designer bioactive molecules.

scholarly journals Uncovering a novel molecular mechanism for scavenging sialic acids in bacteria

2020 ◽  
Vol 295 (40) ◽  
pp. 13724-13736 ◽  
Author(s):  
Andrew Bell ◽  
Emmanuele Severi ◽  
Micah Lee ◽  
Serena Monaco ◽  
Dimitrios Latousakis ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Molecular Mechanism ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
2D Nmr ◽  
Site Directed Mutagenesis ◽  
Bioinformatics Analyses ◽  
E Coli ◽  
Acetylneuraminic Acid ◽  
Protein Dimer

The human gut symbiont Ruminococcus gnavus scavenges host-derived N-acetylneuraminic acid (Neu5Ac) from mucins by converting it to 2,7-anhydro-Neu5Ac. We previously showed that 2,7-anhydro-Neu5Ac is transported into R. gnavus ATCC 29149 before being converted back to Neu5Ac for further metabolic processing. However, the molecular mechanism leading to the conversion of 2,7-anhydro-Neu5Ac to Neu5Ac remained elusive. Using 1D and 2D NMR, we elucidated the multistep enzymatic mechanism of the oxidoreductase (RgNanOx) that leads to the reversible conversion of 2,7-anhydro-Neu5Ac to Neu5Ac through formation of a 4-keto-2-deoxy-2,3-dehydro-N-acetylneuraminic acid intermediate and NAD+ regeneration. The crystal structure of RgNanOx in complex with the NAD+ cofactor showed a protein dimer with a Rossman fold. Guided by the RgNanOx structure, we identified catalytic residues by site-directed mutagenesis. Bioinformatics analyses revealed the presence of RgNanOx homologues across Gram-negative and Gram-positive bacterial species and co-occurrence with sialic acid transporters. We showed by electrospray ionization spray MS that the Escherichia coli homologue YjhC displayed activity against 2,7-anhydro-Neu5Ac and that E. coli could catabolize 2,7-anhydro-Neu5Ac. Differential scanning fluorimetry analyses confirmed the binding of YjhC to the substrates 2,7-anhydro-Neu5Ac and Neu5Ac, as well as to co-factors NAD and NADH. Finally, using E. coli mutants and complementation growth assays, we demonstrated that 2,7-anhydro-Neu5Ac catabolism in E. coli depended on YjhC and on the predicted sialic acid transporter YjhB. These results revealed the molecular mechanisms of 2,7-anhydro-Neu5Ac catabolism across bacterial species and a novel sialic acid transport and catabolism pathway in E. coli.

Macromolecular Targets of Antiparasitic Germacranolide Sesquiterpenoids: An In Silico Investigation

2020 ◽  
Vol 23 (6) ◽  
pp. 477-503
Author(s):  
Phillip M. Arnston ◽  
William N. Setzer
Keyword(s):  
Natural Products ◽  
In Silico ◽  
Density Functional ◽  
Molecular Mechanisms ◽  
Neglected Tropical Diseases ◽  
Treatment Options ◽  
Protein Structures ◽  
Current Treatment ◽  
In Silico Screening ◽  
Protein Targets

Background: The parasitic protozoal infections leishmaniasis, human African trypanosomiasis, and Chagas disease are neglected tropical diseases that pose serious health risks for much of the world’s population. Current treatment options suffer from limitations, but plantderived natural products may provide economically advantageous therapeutic alternatives. Several germacranolide sesquiterpenoids have shown promising antiparasitic activities, but the mechanisms of activity have not been clearly established. Objective: The objective is to use in silico screening of known antiparasitic germacranolides against recognized protozoal protein targets in order to provide insight into the molecular mechanisms of activity of these natural products. Methods: Conformational analyses of the germacranolides were carried out using density functional theory, followed by molecular docking. A total of 88 Leishmania protein structures, 86 T. brucei protein structures, and 50 T. cruzi protein structures were screened against 27 antiparasitic germacranolides. Results: The in-silico screening has revealed which of the protein targets of Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are preferred by the sesquiterpenoid ligands.

Impact of Natural Dietary Agents on Multiple Myeloma Prevention and Treatment: Molecular Insights and Potential for Clinical Translation

2020 ◽  
Vol 27 (2) ◽  
pp. 187-215 ◽  
Author(s):  
Lavinia Raimondi ◽  
Angela De Luca ◽  
Gianluca Giavaresi ◽  
Agnese Barone ◽  
Pierosandro Tagliaferri ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Multiple Myeloma ◽  
Natural Products ◽  
Bone Disease ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Hematologic Malignancy ◽  
Signal Transduction Pathways ◽  
Pharmacologically Active ◽  
Dietary Agents

: Chemoprevention is based on the use of non-toxic, pharmacologically active agents to prevent tumor progression. In this regard, natural dietary agents have been described by the most recent literature as promising tools for controlling onset and progression of malignancies. Extensive research has been so far performed to shed light on the effects of natural products on tumor growth and survival, disclosing the most relevant signal transduction pathways targeted by such compounds. Overall, anti-inflammatory, anti-oxidant and cytotoxic effects of dietary agents on tumor cells are supported either by results from epidemiological or animal studies and even by clinical trials. : Multiple myeloma is a hematologic malignancy characterized by abnormal proliferation of bone marrow plasma cells and subsequent hypercalcemia, renal dysfunction, anemia, or bone disease, which remains incurable despite novel emerging therapeutic strategies. Notably, increasing evidence supports the capability of dietary natural compounds to antagonize multiple myeloma growth in preclinical models of the disease, underscoring their potential as candidate anti-cancer agents. : In this review, we aim at summarizing findings on the anti-tumor activity of dietary natural products, focusing on their molecular mechanisms, which include inhibition of oncogenic signal transduction pathways and/or epigenetic modulating effects, along with their potential clinical applications against multiple myeloma and its related bone disease.

Understanding the molecular mechanism of the stereoselective conversion of N-trialkylsilyloxy nitronates into bicyclic isoxazoline derivatives

2021 ◽  
Author(s):  
Agnieszka Kącka-Zych ◽  
Radomir Jasinski
Keyword(s):  
Density Functional Theory ◽  
Molecular Mechanism ◽  
Electron Density ◽  
Density Functional ◽  
Dft Method ◽  
Functional Theory ◽  
Molecular Electron ◽  
Molecular Electron Density Theory

Conversion of N-trialkylsilyloxy nitronates into bicyclic isoxazoline derivatives has been explored using Density Functional Theory (DFT) method within the context of the Molecular Electron Density Theory (MEDT) at the B97XD(PCM)/6-311G(d,p)...

scholarly journals NMR of Natural Products as Potential Drugs

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3763
Author(s):  
Poul Erik Hansen
Keyword(s):  
Natural Products ◽  
Hydrogen Bonding ◽  
Density Functional ◽  
Chemical Shifts ◽  
Isotope Effects ◽  
Density Functional Theory Calculations ◽  
Intramolecular Hydrogen Bonding ◽  
Chemical Equilibria ◽  
Nmr Techniques ◽  
Intramolecular Hydrogen

This review outlines methods to investigate the structure of natural products with emphasis on intramolecular hydrogen bonding, tautomerism and ionic structures using NMR techniques. The focus is on 1H chemical shifts, isotope effects on chemical shifts and diffusion ordered spectroscopy. In addition, density functional theory calculations are performed to support NMR results. The review demonstrates how hydrogen bonding may lead to specific structures and how chemical equilibria, as well as tautomeric equilibria and ionic structures, can be detected. All these features are important for biological activity and a prerequisite for correct docking experiments and future use as drugs.

scholarly journals Crystal structures of an E1–E2–ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lingmin Yuan ◽  
Zongyang Lv ◽  
Melanie J. Adams ◽  
Shaun K. Olsen
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Molecular Mechanisms ◽  
Conformational State ◽  
Biochemical Data ◽  
Conformational States ◽  
Product Release ◽  
Closed Conformation ◽  
Open Conformation ◽  
Conjugating Enzymes

AbstractE1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1–E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1–E2–Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1–E2–ubiquitin thioester mimetic. Two distinct architectures of the complex are observed, one in which the Ub thioester (Ub(t)) contacts E1 in an open conformation and another in which Ub(t) instead contacts E2 in a drastically different, closed conformation. Altogether our structural and biochemical data suggest that these two conformational states represent snapshots of the E1–E2–Ub complex pre- and post-thioester transfer, and are consistent with a model in which catalysis is enhanced by a Ub(t)-mediated affinity switch that drives the reaction forward by promoting productive complex formation or product release depending on the conformational state.

scholarly journals Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

2020 ◽  
Vol 295 (36) ◽  
pp. 12618-12634
Author(s):  
H. Diessel Duan ◽  
Nishya Mohamed-Raseek ◽  
Anne-Frances Miller
Keyword(s):  
Electron Transfer ◽  
Density Functional ◽  
Protein Denaturation ◽  
Rhodopseudomonas Palustris ◽  
Density Functional Theory Calculations ◽  
Site Directed Mutagenesis ◽  
Functional Theory ◽  
Electron Transfer Flavoprotein ◽  
Midpoint Potential ◽  
Ct Complex

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

Cellular and molecular mechanisms of statins: an update on pleiotropic effects

2015 ◽  
Vol 129 (2) ◽  
pp. 93-105 ◽  
Author(s):  
Mamoru Satoh ◽  
Yuji Takahashi ◽  
Tsuyoshi Tabuchi ◽  
Yoshitaka Minami ◽  
Makiko Tamada ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Chronic Inflammation ◽  
Molecular Mechanism ◽  
Coronary Atherosclerosis ◽  
Molecular Mechanisms ◽  
Cell Injury ◽  
Plaque Instability ◽  
Beneficial Effects ◽  
Reductase Inhibitors ◽  
Artery Disease

Coronary artery disease (CAD) is the leading cause of death worldwide. The efficacy and safety of statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) in primary and secondary prevention of CAD are confirmed in several large studies. It is well known that statins have some pleiotropic, anti-atherosclerotic effects. We review the molecular mechanisms underlying the beneficial effects of statins revealed in recently published studies. Endothelial cell injury is regarded as the classic stimulus for the development of atherosclerotic lesions. In addition, the inflammatory process plays an important role in the aetiology of atherosclerosis. In particular, chronic inflammation plays a key role in coronary artery plaque instability and subsequent occlusive thrombosis. Our previous reports and others have demonstrated beneficial effects of statins on endothelial dysfunction and chronic inflammation in CAD. A better understanding of the molecular mechanism underlying the effectiveness of statins against atherosclerosis may provide a novel therapeutic agent for the treatment of coronary atherosclerosis. The present review summarizes the cellular and molecular mechanism of statins against coronary atherosclerosis.

scholarly journals Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

2016 ◽  
Vol 147 (6) ◽  
pp. 437-449 ◽  
Author(s):  
Petronel Tuluc ◽  
Bruno Benedetti ◽  
Pierre Coste de Bagneaux ◽  
Manfred Grabner ◽  
Bernhard E. Flucher
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Site Directed Mutagenesis ◽  
Control Voltage ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activation Kinetics ◽  
Transmembrane Segments

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

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