Marta Kollárová

Thioredoxin system - a novel therapeutic target.

Nowadays there are numerous pathogens that have created a resistance to commonly used antibiotics and drugs. Therefore research is focused on finding new therapeutic targets and on determination of their 3D structures that could help in designing new effective substances and inhibitors. Thioredoxin system not only plays a crucial role as thiol/disulfide redox controller, it is also essential for certain organisms as the only system ensuring the redox homeostasis. It is the redox-regulating function, which makes thioredoxin and thioredoxin reductase attractive for scientific research, especially in many studies of diseases caused by redox instability. Thanks to these facts, the proteins of thioredoxin system are suitable candidates for new therapeutic purposes. In this review we summarized the basic features of the thioredoxin system, we justified why the proteins of thioredoxin system are appropriate therapeutical targets and we provided overview of the possibilities of their inhibition by several types of inhibitors.

Nuclease-like activity of a cyclic tetrameric schiff base (tetraanhydroaminobenzaldehyde) N-coordinated copper complex

Chemical nucleases are compounds that are able to bind to the surface of nucleic acids and are involved in the generation of free radicals which cleave nucleic acids. The aim of this study was to investigate the interaction of copper(II) tetrabenzo [b,f,j,n] [1,3,9,13] tetraazacyclohexadecine (Cu(TAAB)2+) with DNA and determine the DNase-like activity of this complex. Dichroic properties, electronic absorption and electrophoretical techniques were used to study the reaction of the complex with DNA. Cu(TAAB)2+ affected the dichroic properties and u.v. absorption of calf thymus DNA under anaerobic conditions. The anaerobic melting point of DNA was not affected in the presence of Cu(TAAB)2+. The hyperchromicity of DNA at 260 nm in the presence of Cu(TAAB)2+ with both ascorbic acid and oxygen was determined and compared with the known chemical nuclease Cu(phenantroline)2. Plasmid DNA in the presence of ascorbic acid and oxygen was effectively cleaved by Cu(TAAB)2+ in a concentration dependent manner. It is concluded that the Cu(TAAB)2+ complex is a potential chemical nuclease.

Nuclease mimetic effect of copper complex compounds

The nuclease reaction of a copper complex with the macrocyclic Schiff base ligand tetrabenzo [b,f,j,n][1,3,9,13] tetraazacyclohexadecine (TAAB) at the cleavage of DNA templates has been investigated and compared with that of the copper phenanthroline complex. The labeled single-stranded GC containing 25-mer oligonucleotide, single-stranded AT 20-mer oligonucleotide and double-stranded AT 20-mer oligonucleotide with the complemented sequence in the presence of ascorbic acid and aerobic conditions were used as the substrates. The AT specificity of Cu(TAAB)2+ for both the single-stranded and double-stranded templates may be explained by the specific formation of intercalated complex and oxygen radicals that nick the DNA.

Crystallization and preliminary diffraction studies of malate dehydrogenase from Streptomyces aureofaciens.

Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens was crystallized either in the absence or in the presence of NADH or NADPH coenzymes by hanging-drop vapour-diffusion method. An X-ray study has shown, that MDH crystals belong to space group C222(1) with unit-cell parameters a = 53.2 A, b = 104.6 A, c = 520.0 A, alpha = beta = gamma = 90( degrees ), MDH-NADH crystals to space group C2 with unit-cell parameters a = 51.5 A, b = 51.5 A, c = 256 A, alpha = beta = gamma = 90( degrees ), and MDH-NADPH crystals to space group C222(1) with unit-cell parameters a = 72, A b = 72 A, c = 520 A, alpha = beta = gamma = 90( degrees ). The crystal of native MDH diffracted to 2.1 A resolution.

Purification and partial characterization of two thioredoxins from Streptomyces aureofaciens

Thioredoxins are low molecular weight proteins, which participate in a wide spectrum of biochemical reactions. Two thioredoxins from Streptomyces aureofaciens 3239 have been purified to homogeneity by a sequence of chromatography steps including chromatography on Sephacryl S‐300, Phenyl Sepharose CL 4B and MonoQ HR 5/5. Thioredoxin activity clearly separates into two protein fractions on MonoQ HR 5/5 chromatography. Molecular weights determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed Mr ∼ 10,500 for thioredoxin 1 (TR1) and Mr ∼ 11,000 for thioredoxin 2 (TR2). The isoelectric points of the two thioredoxins are different pI = 4.7 for TR1 and 5.6 for TR2, respectively. Both were effectively reduced with NADPH in reaction catalyzed by Streptomyces aureofaciens thioredoxin reductase. The specific activity of viewly for discovered TR2 is about 1/4 of the specific activity of TR1. Both thioredoxins activate spinach NADPH‐malate dehydrogenase. Activation of this enzyme by TR2 is only half effective than by TR1. The stability of TR1 is high and similar to thioredoxins from other organisms unlike the activity of TR2 which is decreassed during purification. The proteins diversed in their contents in exponentialy growing mycelium.

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.

Crystallization and diffraction analysis of thioredoxin reductase from Streptomyces coelicolor.

Thioredoxin reductases are homodimeric flavoenzymes that catalyze the transfer of electrons from NADPH to oxidized thioredoxin substrate. Bacterial thioredoxin reductases represent a promising target for the development of new antibiotics. Recombinant thioredoxin reductase TrxB from Streptomyces coelicolor was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected from cryocooled crystals to 2.4 Å resolution using a synchrotron-radiation source. The crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 82.9, b = 60.6, c = 135.4 Å, α = γ = 90.0, β = 96.5°.

Expression, purification and X-ray crystallographic analysis of thioredoxin from Streptomyces coelicolor

Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases. In turn, they are reduced by electrons obtained from the NADPH-containing thioredoxin reductase. Thioredoxins have been isolated and characterized from a large number of organisms. The Gram-positive bacterium Streptomyces coelicolor contains three thioredoxins that are involved in unknown biological processes. trxA from S. coelicolor was cloned and expressed in Escherichia coli and the protein purified and crystallized using the hanging-drop method of vapour diffusion. The crystal structure of thioredoxin A has been determined at 1.5 A resolution using a synchrotron-radiation source. The protein reveals a thioredoxin-like fold with a typical CXXC active site. The crystal exhibits the symmetry of space group P2(1)2(1)2, with unit-cell parameters a = 43.6, b = 71.8, c = 33.2 A.

PURIFICATION AND PARTIAL CHARACTERIZATION OF THIOREDOXIN REDUCTASE FROM Streptomyces aureofaciens

Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide‐containing substrate. Thioredoxin reductase from Streptomyces aureofaciens 3239 has been purified to homogeneity by a two‐step chromatographic procedure including anion‐exchange chromatography and affinity chromatography on 2′5′‐ADP‐Sepharose 4B. Molar mass determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed 69 kDa for the native protein and 34.8 kDa for the enzyme subunit. The isoelectric point determined by isoelectric focusing gel electrophoresis was 4.3. TrxR effectively catalyzed the reduction of DTNB in the presence of S. aureofaciens thioredoxin‐1. TrxR activity in the presence of S. aureofaciens thioredoxin‐2 was only 1/4 of the activity with thioredoxin‐1 (1). The activity of pure TrxR decreased drastically in the presence of NADPH, while NADP+ as well as Streptomyces aureofaciens thioredoxin‐1 protected the enzyme from inactivation. These results indicate that thioredoxin reductase activity in bacteria could be modulated by the redox status of NADP+/NADPH and thioredoxin pools.

PCR cloning and sequencing of the coding portion of the thioredoxin- encoding gene from Streptomyces aureofaciens BMK*

The nucleotide sequence of the PCR-cloned coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK was determined. The deduced 106-amino-acid sequence was compared with three other thioredoxins.