Katarzyna Ostrowska

Modulation of thiamine metabolism in Zea mays seedlings under conditions of abiotic stress

The responses of plants to abiotic stress involve the up-regulation of numerous metabolic pathways, including several major routes that engage thiamine diphosphate (TDP)-dependent enzymes. This suggests that the metabolism of thiamine (vitamin B1) and its phosphate esters in plants may be modulated under various stress conditions. In the present study, Zea mays seedlings were used as a model system to analyse for any relation between the plant response to abiotic stress and the properties of thiamine biosynthesis and activation. Conditions of drought, high salt, and oxidative stress were induced by polyethylene glycol, sodium chloride, and hydrogen peroxide, respectively. The expected increases in the abscisic acid levels and in the activities of antioxidant enzymes including catalase, ascorbate peroxidase, and glutathione reductase were found under each stress condition. The total thiamine compound content in the maize seedling leaves increased under each stress condition applied, with the strongest effects on these levels observed under the oxidative stress treatment. This increase was also found to be associated with changes in the relative distribution of free thiamine, thiamine monophosphate (TMP), and TDP. Surprisingly, the activity of the thiamine synthesizing enzyme, TMP synthase, responded poorly to abiotic stress, in contrast to the significant enhancement found for the activities of the TDP synthesizing enzyme, thiamine pyrophosphokinase, and a number of the TDP/TMP phosphatases. Finally, a moderate increase in the activity of transketolase, one of the major TDP-dependent enzymes, was detectable under conditions of salt and oxidative stress. These findings suggest a role of thiamine metabolism in the plant response to environmental stress.

Molecular characterization of the thi3 gene involved in thiamine biosynthesis in Zea mays: cDNA sequence and enzymatic and structural properties of the recombinant bifunctional protein with 4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate) kinase and thiamine monophosphate synthase activities

A thiamine biosynthesis gene, thi3, from maize Zea mays has been identified through cloning and sequencing of cDNA and heterologous overexpression of the encoded protein, THI3, in Escherichia coli. The recombinant THI3 protein was purified to homogeneity and shown to possess two essentially different enzymatic activities of HMP(-P) [4-amino-5-hydroxymethyl-2-methylpyrimidine (phosphate)] kinase and TMP (thiamine monophosphate) synthase. Both activities were characterized in terms of basic kinetic constants, with interesting findings that TMP synthase is uncompetitively inhibited by excess of one of the substrates [HMP-PP (HMP diphosphate)] and ATP. A bioinformatic analysis of the THI3 sequence suggested that these activities were located in two distinct, N-terminal kinase and C-terminal synthase, domains. Models of the overall folds of THI3 domains and the arrangements of active centre residues were obtained with the SWISS-MODEL protein modelling server, on the basis of the known three-dimensional structures of Salmonella enterica serotype Typhimurium HMP(-P) kinase and Bacillus subtilis TMP synthase. The essential roles of Gln98 and Met134 residues for HMP kinase activity and of Ser444 for TMP synthase activity were experimentally confirmed by site-directed mutagenesis.

Chelate Ring Size Effect as a Factor of Selective Fluorescent Recognition of Zn(2+) Ions by Pyrrolo[2,3-b]quinoxaline with a Substituted 2-Pyridyl Group Receptor.

Analysis of the spectral properties and structural differences of two turn-on ratiometric fluorescent receptors for Zn(2+) and Cd(2+) ions, derivatives of pyrrolo[2,3-b]quinoxaline (2), and earlier published 3 (Ostrowska et al. CrystEngComm 2015, 17, 498-502) was performed. Both ligands are E/Z push-pull olefins interconverting at room temperature, with barriers to rotation about enamine double bonds, from E to Z isomers of 19.3 ± 0.1 and 16.9 ± 0.3 kcal/mol and from Z to E of 16.9 ± 0.3 and 15.7 ± 0.2 kcal/mol, respectively. Diastereoisomers (E)-2 and (Z)-2 were isolated and characterized by X-ray structural analysis. The formation of complexes by (E/Z)-2 with acetates and acetylacetonates of Zn(2+) and Cd(2+) was monitored by UV-vis, fluorescence, and (1)H NMR titrations in acetonitrile, respectively. X-ray structural analysis for isolated [(E)-2]2Zn in relation to earlier published (E)-3-ZnOAc revealed the formation of a six-coordinated zinc ion with six- and four-membered bis-chelate rings by (E)-2. The chelate effect increases the ligand affinity for Zn(2+) (log β12 = 12.45) and causes the elongation of nitrogen-metal bonds. Extension of the coordination cavity size allows coordination of a cadmium ion. The introduction of a flexible ethylene linker between the fluorophore and ionophore pyridyl groups in 3 significantly affects the selectivity of zinc-ion recognition. The distorted tetrahedral geometry of (E)-3-ZnOAc with a four-coordinated zinc ion appears to be the most preferred because of the short donor-zinc distance with a 1:1 binding mode. The formation of the small coordination cavity size with six-membered bis-chelate rings provides an effective overlap of zinc and donor orbitals, precluding the coordination of a cadmium ion in the same manner as zinc.

Polypeptide components of oligomeric legumin-like thiamin-binding protein from buckwheat seeds characterized by partial amino acid sequencing and photoaffinity labeling

Among thiamin-binding proteins that ubiquitously occur in plant seeds, that of common buckwheat became a model of extensive studies of the chemical mechanism of ligand-protein interaction. In this work, the polypeptide components of buckwheat seed thiamin-binding protein (BSTBP) are identified and characterized. We suggest that BSTBP is probably a fraction of major storage 13 S globulin (legumin), has an average molecular mass of 235 kDa and comprises hexamers of 57-kDa and 38-kDa subunits in variable combinations. Each subunit is a pair of disulfide-linked polypeptide chains, 36 kDa plus 24 kDa and two-times 22 kDa, respectively. The N-terminal sequences of 22-kDa and 24-kDa components show strict homology with those reported for “basic subunits” of buckwheat legumin. By photoaffinity labeling of BSTBP with 4-azido-2-nitrobenzoylthiamine, it is shown that the 36-kDa chain plays the major role in thiamin binding, but the other chains may also be variably involved. Putative thiamin-binding fragments are identified and sequenced.

SYNTHESIS AND TRANSAMINATION OF ENAMINONES : DERIVATIVES OF 1-PHENYL-4-(PHENYLHYDROXYMETHYLIDENE)-PYRROLIDINE-2,3,5-TRIONE

Summary. The reaction of 1-phenyl-4-(phenylhydroxymethylidene)-pyrrolidine-2,3,5-trione with difunctional bases (α-,β-,γ-amino acid derivatives or aminoethanol) leads to a mixture of tautomeric Schiff bases and enaminones. Some of the products easily undergo transamination at their enaminone moiety.Zusammenfassung. Die Reaktion von 1-Phenyl-4-(phenylhydroxymethyliden)pyrrolidin-2,3,5-trions mit difunktionellen Basen (α-,β-γ-Aminosäurederivativen oder Aminoethanol) führt zu einem Gemisch der tautomeren Schiffschen Basen und Enaminone. Einige dieser Verbindungen werden leicht am Enaminonfragment transaminiert.

Pyrrolo[2,3-b]quinoxaline with 2-(2-aminoethyl)pyridine chain highly selective fluorescent receptor for Zn2+ exhibiting a dual fluorescence and AIEE in crystalline state

This article describes the synthesis of a selective dual emissive intramolecular charge transfer (ICT) receptor for zinc ion in acetonitrile, exhibiting aggregation induced emission enhancement (AIEE) in the solid state due to the formation of inter- or intra-molecular hydrogen bonds and aromatic donor–acceptor interaction with H- or J-aggregation.

Synthesis and NMR Structural Assignment of 3-Benzoylpyrrolo-[2,3-b]quinoxalin-2(4H)-ones

A series of 3-benzoylpyrrolo[2,3-b]quinoxalin-2(4H)-ones, potential bioactive compounds have been synthesized. Their preparation is based on the efficient, regiospecific condensation of o-phenylenediamine with pyrrolidine-2,3,5-trione (1) or its enaminone derivatives, respectively, affording two polymorphic forms of the latter in solid. The NMR spectral assignment of 3-benzoylpyrrolo[2,3-b]quinoxalin-2(4H)-ones confirms the presence of only one isomer with enaminone moiety in solution.

Ratiometric fluorescent Zn2+ and In3+ receptors of fused pyrazine with an aminopropanol chain in acetonitrile

A series of new potential intramolecular charge transfer ICT fluorescent receptors of Zn2+, Cd2+, Hg2+, Ga3+, In3+, and Tl3+ ions based on N-aryl or N-alkyl variously fused pyridopyrrolopyrazine or pyrrolo[2,3-b]quinoxaline with an integrated donor group, such as 3-aminopropanol, were synthesized and verified. Among [N, N, O] tridentate donors 4a, 6a–d, 10a, 12e, and 14e, only the integrated fluorophore-receptors with the nitrogen atom at the N-5 position of heterocyclic systems, pyrido[2,3-b]pyrrolo[2,3-e]pyrazine 6a, selectively respond to zinc and indium with significant fluorescent enhancement and different mechanisms of binding. The first example of Z to E interconversion of enaminone forms of a ligand responsible for Zn2+ complex fluorescence enhancement was documented.

THE REGIOSELECTIVE SYNTHESIS OF 2H-PYRIDO [2,3-b] PYRROLO [2,3-e] PYRAZIN-2-ONE

- The regioselective reaction of l-aryl-4-(phenylhydroxymethylidene)-pyrrolidine-2,3,5-triones 1 with 2,3-diaminopyridine in boiling glacial AcOH in the presence of TsOH led to the formation of new 2H-pyrido[2,3-b]pyrrolo[2,3-e]pyrazin-2-ones. The structure of this fused heterocyclic system was confirmed by IR, 1D and 2D NMR experiments.

Application of Bertocco-Yoshida interpolated DFT algorithm to NMR data analysis

The paper presents a new application of the Bertocco-Yoshida order 1 (BY1) interpolated discrete Fourier transform (IpDFT) with leakage correction (LC) to analysis of nuclear magnetic resonance (NMR) signals. It is shown how the BY-LC IpDFT algorithm can be used for NMR spectrum quantification, i.e. 1) estimation of frequencies, damping factors (relaxation times), amplitudes and phases of signals components of interest, 2) phase correction of the whole spectrum or its selected fragment, 3) fast calculation of a reliable initial starting point for advanced optimization-based phase correction algorithms. In the paper synthetic NMR-like and real NMR signals are analyzed as examples.