Structural peculiarities of tandem repeats and their clinical significance.

While it is well established that a mere 2% of human DNA nucleotides are involved in protein coding, the remainder of the DNA plays a vital role in the preservation of normal cellular genetic function. A significant proportion of tandem repeats (TRs) are present in non-coding DNA. TRs - specific sequences of nucleotides that entail numerous repetitions of a given fragment. In this study, we employed our novel algorithm grounded in finite automata theory, which we refer to as Dafna, to investigate for the first time the likelihood of these nucleotide sequences forming non-canonical DNA structures (NS). Such structures include G-quadruplexes, i-motifs, hairpins, and triplexes. The tandem repeats under consideration in our research encompassed sequences containing 1 to 6 nucleotides per repeated fragment. For comparison, we employed a set of randomly generated sequences of the same length (60 nucleotides) as a benchmark. The outcomes of our research exposed a disparity between the potential for NS formation in random sequences and tandem repeats. Our findings affirm that the propensity of DNA and RNA to form NS is closely tied to various genetic disorders, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. In the concluding discussion, we present a proposal for a new therapeutic mechanism to address these diseases. This novel approach revolves around the ability of specific nucleic acid fragments to form multiple types of NS.

The crystal structure of yeast mitochondrial type pyrophosphatase provides a model to study pathological mutations in its human ortholog.

Mutations in human ppa2 gene encoding mitochondrial inorganic pyrophosphatase (PPA2) result in the mitochondria malfunction in heart and brain and lead to early death. In comparison with its cytosolic counterpart, PPA2 of any species is a poorly characterized enzyme with a previously unknown 3D structure. We report here the crystal structure of PPA2 from yeast Ogataea parapolymorpha (OpPPA2), as well as its biochemical characterization. OpPPA2 is a dimer, demonstrating the fold typical for other eukaryotic Family I pyrophosphatases, including the human cytosolic enzyme. Cofactor Mg2+ ions found in OpPPA2 structure have similar coordination to most known Family I pyrophosphatases. Most of the residues associated with the pathological mutations in human PPA2 are conserved in OpPPA2, and their structural context suggests possible explanations for the effects of the mutations on the human enzyme. In this work, the mutant variant of OpPPA2, Met52Val, corresponding to the natural pathogenic variant Met94Val of human PPA2, is characterized. The obtained structural and biochemical data provide a step to understanding the structural basis of PPA2-associated pathologies.

Multifunctionality of arginine residues in the active sites of non-canonical d-amino acid transaminases.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications.

Proton Sponge Zwitterions with Positively and Negatively Charged Hydrogen Bonds: Extremely Polar Organic Compounds and Interplay of [NHN]+ and [NHN]- Properties.

Six previously unknown zwitterions with positively and negatively charged [NHN] hydrogen bonds were synthesized by acylation of 4,5-bis(dimethylamino)-1-tosylamino-8-aminonaphthalene with subsequent alkaline treatment of the resulting 8-acylamino derivatives. Using NMR and XRD measurements in conjunction with quantum chemical DFT/PBE1PBE/6-311++G(d,p) calculations, it was shown that the negatively charged [NHN]- bond in such compounds commonly differs from the [NHN]+ bond by significantly lower linearity, higher asymmetry, and moderate to strong paramagnetic shift of the chelated NH proton signal. Among other remarkable findings, the most important are (1) unusually high polarity (µ = 21-26 D) of the obtained zwitterions, (2) sharp difference in structures of the solid 1,8-bis(tosylated) zwitterion (BTZ) grown from MeCN or DMF, and (3) registration for one of the stereoisomers of BTZ with the record short [NHN]- hydrogen bridge (N···N = 2.510 Å) almost reaching the theoretical limit (2.50 Å) for the [NHN]+ hydrogen bond.

Tridentate Nitrogen Ligand as a Tool for the Construction of Well-Defined Rare Earth Trichloride Complexes.

LnCl3(THF)3 (Ln = Y, La ÷ Nd, Sm ÷ Lu) readily react with the tridentate 1,3,5-trimethyl-1,3,5-triazacyclohexane (Me3tach) ligand to form mono- or binuclear lanthanide trichloride complexes, depending on the stoichiometry of the reaction and the ionic radius of the metal: mononuclear pseudosandwich [LnCl3(Me3tach)2], (Ln = Y, La ÷ Ho) or binuclear complexes [Ln2Cl6(Me3tach)3], or [LnCl3(Me3tach)(THF)]2 (Ln = Sm, Tb). Detailed analysis of the NMR data of [LnCl3(Me3tach)2] complexes with paramagnetic lanthanide ions showed that their structures remained unchanged in the toluene solution. A series of isomorphous complexes [LnCl3(Me3tach)(Py)2] (Ln = La, Sm, Tb, Er, Lu; Py = pyridine) have been obtained by the recrystallization of either mononuclear or binuclear complexes from pyridine. Complexes of terbium and europium ions with the Me3tach ligand exhibit relatively high quantum yields of metal-centered luminescence (0.39 and 0.32, respectively).

CASE-DFT Structure Elucidation of Proton-Deficient Chlorodepsidones from the Indonesian Lichen Teloschistes flavicans and Structure Revision of Flavicansone.

Biodiscovery efforts in Indonesia have aimed to explore the understudied chemical diversity of its rich lichen flora, seeking to find new products endowed with significant biological properties. The chemical screening of a Teloschistes flavicans extract led to selection of this species for further investigation. LC/MS and 1H NMR-based dereplication pinpointed six chlorodepsidones from the thallus of a sample of this lichen. This led to the streamlined isolation and the subsequent structure elucidation of the three new compounds norflavicansone 1, flavicansone 2, and isocaloploicin 3, along with the known chlorodepsidones 4-6, stictic acid 7, aurantiamide acetate 8, and parietin 9. The challenging structure elucidation of these proton-deficient metabolites benefited from a state-of-the-art workflow involving a synergistic combination of Computer-Assisted Structure Elucidation (CASE) and Density Functional Theory (DFT) calculations of the top-ranked candidates. This investigation also led to the revision of flavicansone's structure, previously described from this species. The three new molecules that are being reported here are remarkable in that they represent hybrid depsidones in which one of the aromatic rings is derived from orsellinic acid and the other is derived from ß-orcinol, a rare structural feature for lichen depsidones.

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

Mild and efficient synthesis and base-promoted rearrangement of novel isoxazolo[4,5-b]pyridines.

An efficient method for the synthesis of isoxazolo[4,5-b]pyridines has been developed on the basis of readily available 2-chloro-3-nitropyridines via the intramolecular nucleophilic substitution of the nitro group as a key step. The previously unknown base-promoted Boulton-Katritzky rearrangement of isoxazolo[4,5-b]pyridine-3-carbaldehyde arylhydrazones into 3-hydroxy-2-(2-aryl[1,2,3]triazol-4-yl)pyridines was observed.

Terpene-Containing Analogues of Glitazars as Potential Therapeutic Agents for Metabolic Syndrome.

Metabolic syndrome is a complex of abnormalities involving impaired glucose and lipid metabolism, which needs effective pharmacotherapy. One way to reduce lipid and glucose levels associated with this pathology is the simultaneous activation of nuclear PPAR-alpha and gamma. For this purpose, we synthesized a number of potential agonists based on the pharmacophore fragment of glitazars with the inclusion of mono- or diterpenic moiety in the molecular structure. The study of their pharmacological activity in mice with obesity and type 2 diabetes mellitus (C57Bl/6Ay) revealed one substance that was capable of reducing the triglyceride levels in the liver and adipose tissue of mice by enhancing their catabolism and expressing a hypoglycemic effect connected with the sensitization of mice tissue to insulin. It has also been shown to have no toxic effects on the liver.

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs' regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

Synthesis of N,N'-Unsymmetrical 9-Amino-5,7-dimethyl-bispidines.

A new approach to the preparation of N,N'-unsymmetrically substituted 9-aminobispidines through aminal bridge removal reaction has been developed, the principal feature of which is the ability to selectively functionalize all three nitrogen atoms. The intermediates of 1,3-diazaadamantane's aminal bridge removal reaction are characterized, and the mechanism of this reaction is proposed based on the analysis of their structures. Representatives of the previously unknown saturated heterocyclic 1,5,9-triazatricyclo[5.3.1.03,8]undecane system were obtained and structurally characterized. Thus, it was possible for the first time to obtain 3,7,9-trisubstituted bispidines containing acetyl, Boc, and benzyl groups at nitrogen atoms, which can be independently removed (orthogonal protective groups).

Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure.

The ability to evaluate sperm at the microscopic level, at high-throughput, would be useful for assisted reproductive technologies (ARTs), as it can allow specific selection of sperm cells for in vitro fertilization (IVF). The tradeoff between intrinsic imaging and external contrast agents is particularly acute in reproductive medicine. The use of fluorescence labels has enabled new cell-sorting strategies and given new insights into developmental biology. Nevertheless, using extrinsic contrast agents is often too invasive for routine clinical operation. Raising questions about cell viability, especially for single-cell selection, clinicians prefer intrinsic contrast in the form of phase-contrast, differential-interference contrast, or Hoffman modulation contrast. While such instruments are nondestructive, the resulting image suffers from a lack of specificity. In this work, we provide a template to circumvent the tradeoff between cell viability and specificity by combining high-sensitivity phase imaging with deep learning. In order to introduce specificity to label-free images, we trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation.

Straightforward and Efficient Protocol for the Synthesis of Pyrazolo [4,3-b]pyridines and Indazoles.

An efficient method for the synthesis of pyrazolo [4,3-b]pyridines has been developed on the basis of readily available 2-chloro-3-nitropyridines via a sequence of SNAr and modified Japp-Klingemann reactions. The method offers a number of advantages including utilization of stable arenediazonium tosylates, operational simplicity as well as combining the azo-coupling, deacylation and pyrazole ring annulation steps in a one-pot manner. An unusual rearrangement (C-N-migration of the acetyl group) was observed and a plausible mechanism was proposed based on the isolated intermediates and NMR experiments. In addition, the developed protocol was successfully applied to the synthesis of 1-arylindazoles combining the Japp-Klingemann reaction and cyclization of the resulting hydrazone as a one-pot procedure.

Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures.

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the ß-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

Structure-Functional Examination of Novel Ribonucleoside Hydrolase C (RihC) from Limosilactobacillus reuteri LR1.

Ribonucleoside hydrolase C (RihC, EC 3.2.2.1, 3.2.2.2, 3.2.2.3, 3.2.2.7, 3.2.2.8) belongs to the family of ribonucleoside hydrolases Rih and catalyzes the cleavage of ribonucleosides to nitrogenous bases and ribose. RihC is one of the enzymes that are synthesized by lactobacilli in response to the presence of Klebsiella. To characterize this protein from Limosilactobacillus reuteri LR1, we cloned and expressed it. The activity of the enzyme was studied towards a wide range of substrates, including ribonucleosides, deoxyribonucleosides as well as an arabinoside. It was shown that the enzyme is active only with ribonucleosides and arabinoside, with the best substrate being uridine. The thermal stability of this enzyme was studied, and its crystal structure was obtained, which demonstrated the tetrameric architecture of the enzyme and allowed to shed light on a correlation between its structure and enzymatic activity. Comprehensive comparisons of all known RihC structures, both existing crystal structures and computed model structures from various species, were made, allowing for the identification of structural motifs important for enzyme functioning.

Ominoxanthone-The First Xanthone Linearly Fused to a γ-Lactone from Cortinarius ominosus Bidaud Basidiomata. CASE- and DFT-Based Structure Elucidation.

The UHPLC-HRMS analysis of Cortinarius ominosus basidiomata extract revealed that this mushroom accumulated elevated yields of an unreported specialized metabolite. The molecular formula of this unknown compound, C17H10O8, indicated that a challenging structure elucidation lay ahead, owing to its critically low H/C atom ratio. The structure of this new isolate, namely ominoxanthone (1), could not be solved from the interpretation of the usual set of 1D/2D NMR data that conveyed too limited information to afford a single, unambiguous structure. To remedy this, a Computer-Assisted Structure Elucidation (CASE) workflow was used to rank the different possible structure candidates consistent with our scarce spectroscopic data. DFT-based chemical shift calculations on a limited set of top-ranked structures further ascertained the determined structure for ominoxanthone. Although the determined scaffold of ominoxanthone is unprecedented as a natural product, a plausible biosynthetic scenario involving a precursor known from cortinariaceous sources and classical biogenetic reactions could be proposed.

To the Understanding of Catalysis by D-Amino Acid Transaminases: A Case Study of the Enzyme from Aminobacterium colombiense.

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are highly efficient biocatalysts for stereoselective amination. D-amino acid transaminases can catalyze stereoselective transamination producing optically pure D-amino acids. The knowledge of substrate binding mode and substrate differentiation mechanism in D-amino acid transaminases comes down to the analysis of the transaminase from Bacillus subtilis. However, at least two groups of D-amino acid transaminases differing in the active site organization are known today. Here, we present a detailed study of D-amino acid transaminase from the gram-negative bacterium Aminobacterium colombiense with a substrate binding mode different from that for the transaminase from B. subtilis. We study the enzyme using kinetic analysis, molecular modeling, and structural analysis of holoenzyme and its complex with D-glutamate. We compare the multipoint binding of D-glutamate with the binding of other substrates, D-aspartate and D-ornithine. QM/MM MD simulation reveals that the substrate can act as a base and its proton can be transferred from the amino group to the α-carboxylate group. This process occurs simultaneously with the nucleophilic attack of the PLP carbon atom by the nitrogen atom of the substrate forming gem-diamine at the transimination step. This explains the absence of the catalytic activity toward (R)-amines that lack an α-carboxylate group. The obtained results clarify another substrate binding mode in D-amino acid transaminases and underpinned the substrate activation mechanism.

Synthesis of substituted 8H-benzo[h]pyrano[2,3-f]quinazolin-8-ones via photochemical 6π-electrocyclization of pyrimidines containing an allomaltol fragment.

For the first time, we elaborated a method for the synthesis of pyrimidines containing an allomaltol unit. The suggested approach is based on the reaction of 2-(1-(dimethylamino)-3-oxo-3-arylprop-1-en-2-yl)-3-hydroxy-6-methyl-4H-pyran-4-ones with cyanamide. The photochemical behavior of the obtained pyrimidines was investigated. It was shown that for the hydroxy derivatives the main pathway of phototransformation is a 6π-electrocyclization of the 1,3,5-hexatriene system and subsequent [1,9]-H sigmatropic shift leading to dihydrobenzo[h]pyrano[2,3-f]quinazolines. At the same time, for methylated analogues the photoreaction proceeds in two directions resulting in the formation of a mixture of the corresponding dihydrobenzo[h]pyrano[2,3-f]quinazolines and polyaromatic products. The obtained dihydro derivatives are stable compounds and do not undergo aromatization upon further UV irradiation. The structures of two of the dihydrobenzo[h]pyrano[2,3-f]quinazolines were confirmed by X-ray diffraction analysis. Based on the performed studies, a two-stage telescopic method for the synthesis of polyaromatic benzo[h]pyrano[2,3-f]quinazolines including the initial photocyclization of the starting pyrimidines and the final dehydration was proposed.

Mechanisms of nitric oxide generation in living systems.

In modern chemical and biochemical studies, special attention is paid to molecular systems capable of generating nitric oxide (NO), which is one of the most important signalling molecules in the body and can trigger a whole cascade of reactions. Despite the importance of this molecule, the mechanisms of its formation in living organisms remain a subject of debate. This review combines the most important methods of releasing NO from endogenous and exogenous sources. The history of endogenous NO donors dates back more than 150 years, since the synthesis of nitroglycerin, which remains the standard vasodilator today, even though it is known that it and many other similar compounds lead to the development of a nitrate tolerance. Particular awareness is devoted to the mechanisms of NO formation without the participation of enzymes, since these methods are most important for creating exogenous sources of NO as drugs. The study of NO formation methods is centred on both the creation of new NO donors and understanding the mechanisms of tolerance to them.