Search

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

Biochemical and structural properties of three recombinant (R), highly homologous, plant bifunctional nucleases from tomato (R-TBN1), hop (R-HBN1) and Arabis brassica (R-ABN1) were determined. These nucleases cleave single- and double-stranded substrates, as well as both RNA and DNA with nearly the same efficiency. In addition, they are able to cleave several artificial substrates and highly stable viroid RNA. They also possess 3'-nucleotidase activity; therefore, they can be classified as nuclease I family members. Interestingly, poly(G) is resistant to cleavage and moreover it inhibits dsDNase, ssDNase and RNase activity of the studied nucleases. All three nucleases exhibit zinc-dependence and a strong stimulatory effect of Zn²+ for dsDNA cleavage. 3-D models, predicted on the basis of experimental structure of P1 nuclease, show nine amino acid residues responsible for interactions with zinc atoms, located in the same positions as in P1 nuclease. It was also shown that R-TBN1, R-HBN1, and R-ABN1 are all N-glycosylated. Oligosaccharidic chains constitute about 16% of their MW. In addition, an anticancer potential of the R-ABN1 is compared in this work with previously tested R-TBN1, and R-HBN1. R-ABN1 injected intravenously showed 70% inhibitory effect on growth of human prostate carcinoma in athymic mice., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.