THIOREDOXIN SYSTEM OF STREPTOMYCES COELICOLOR : OVEREXPRESSION, ISOLATION, CRYSTALLIZATION AND CHARACTERIZATION

P. Štefanková ¹, J. Maderová ^1,2, M. Kollárová¹, I. Barák ³, Z. Otwinowski²

¹Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 842 15 Bratislava, Slovak Republic

²Department of Biochemistry, Southwestern Medical Center at Dallas, University of Texas, 5323 Harry Hines Blvd., 75 390 Dallas, Texas, USA

³Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta, 842 51 Bratislava, Slovak Republic

Thioredoxin, thioredoxin reductase and coenzyme NADPH together form thioredoxin system, which is ubiquitous from Archaea to man. Thioredoxins (Trx) are small and very stable redox-active proteins containing a reducible disulfide bridge with amino acid sequence Cys-Gly-Pro-Cys. Trx is reduced by electrons from NADPH via thioredoxin reductase (TrxR), flavoprotein homodimeric enzyme [1]. Reduced thioredoxin acts as a major ubiquitous disulfide reductase responsible for maintaining proteins in their reduced state. Tripeptide glutathione also can act to preserve the thiol-disulfide status of proteins in the cell in a manner similar to thioredoxin. However, glutathione is not made by actinomycetes, including streptomycetes and mycobacteria, which instead contain a low-molecular-weight sugar-containing monothiol called mycothiol [2]. So, Streptomyces are suitable model for study of thioredoxin system.

S. coelicolor is a representative of a group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. The 8.667.507 base pair linear chromosome of this organism contains the largest number of genes so far discovered in bacterium [3]. The complete genome sequence of S. coelicolor revealed several possible thioredoxin genes, and thus Streptomyces seems to have a more complex redox system in comparison with other bacterial species.

The presented work describes the overexpression, purification and partial characterization of thioredoxinA and thioredoxin reductase from S. coelicolor. The genes encoding trxA and TrxR were amplified by PCR and inserted into a pET expression vector and used to transform Escherichia coli. TrxA and TrxR were overexpressed by induction with IPTG as hexahistidine fusion proteins and were recovered from a cytoplasm as soluble and active proteins. Homogenous proteins were used for investigation of protein stability, their role in the redox control in the cell and study of protein-protein interaction. Thioredoxin was crystallized using the hanging drop method of vapour diffusion. We have determined the crystal structure of TrxA at 1.5 Å resolution, using a synchrotron radiation source. The protein reveals a thioredoxin-like fold with typical CXXC active site. The crystal exhibits the symmetry of space group P2₁2₁2 with unit cell dimensions 43.6 Å, 71.8Å, 33.2 Å.

Acknowledgements. This work was supported by the research grants from the Slovak Grant Agency VEGA No. 1/0035/03.

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