Structure of multifunctional plant nuclease TBN1

 

T. Kovaľ1, J. Stránský1, 2, T. Podzimek3, P. Lipovová3, J. Matoušek4, K. Fejfarová1,
P. Kolenko1, J. Dušková1, T. Skálová1, J. Hašek1 and J. Dohnálek1

 

1Institute of Macromolecular Chemistry, AS CR, v.v.i., Heyrovského nám. 2, 162 06 Praha 6, Czech Republic

2Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University, Břehová 7, 115 19

Praha 1, Czech Republic

3Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Czech Republic

4Institute of Plant Molecular Biology, Biology Centre, AS CR, v.v.i., Branišovská 31, 370 05 České Budějovice, Czech Republic

koval.tomas@gmail.com

 

Plant nuclease TBN1 (UniProt sequence accession no. Q0KFV0) from Solanum lycopersicum (tomato) is a Zn2+- dependent glycoprotein with a molecular mass of 31.6 kDa (about 37 kDa when glycosylated). TBN1 belongs to plant nuclease I family and plays an important role in specific apoptotic functions, vascular system development, stress response and tissue differentiation in plants [1]. In addition, TBN1 exhibits anticancerogenic properties [2].

Two recombinantly expressed variants of TBN1 (wild type and hypoglycosylated mutant N211D) were used in our study. Datasets for structural analysis were collected at the synchrotron radiation source BESSY II (Helmholtz-Zentrum Berlin). Presence of zinc in the protein was confirmed by X-ray fluorescence and an absorption edge scan. The phase problem was solved using combination of MAD phasing and molecular replacement [3, 4]. 

TBN1 is mainly α-helical with a trinuclear Zn2+ cluster placed in the active site in the center of the wide groove. Three oligosaccharides bonded on the surface serve primarily as a shielding of the hydrophobic regions and therefore contribute to solubility and stability of the enzyme (Figure 1). TBN1 acts as phosphodiesterase cleaving the bond between phosphorus and 3’ hydroxyl group in both single stranded and double stranded forms of DNA and RNA and shows 3’-nucleotidase activity. Moreover, phospholipase C‑like activity of TBN1 was detected using artificial and native substrates for bacterial phospholipase C. Hydrolysis of the phosphodiester bond is caused by a nucleophilic attack of the activated water (hydroxide) molecule followed by creation of penta-coordinated transition state and its breakup into the products.

Figure 1 Fold, secondary structure and main features of TBN1 [5].

 

The work on this project was supported by the Czech Science Foundation, projects no. P302/11/0855, 202/06/0757 and 521/09/1214, by the EC under ELISA grant agreement number 226716 (synchrotron access, projects 09.2.90262 and 10.1.91347), by the Institution research plan AV0Z50510513 of the Institute of Plant Molecular Biology, Biology Centre. We acknowledge support of the Ministry of Education, Youth and Sports of the Czech Republic (grant No. CZ.1.07/2.3.00/30.0029). The authors wish to thank Dr. U. Müller of the Helmholtz-Zentrum Berlin, for support at the beam line.

 

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