MS09-P12 Structural insights into an evolutionary intermediate of dUTPase Beata Vertessy (BME ABÉT Budapest University of Technology and Economics, Budapest, Hungary) Andras Benedek (BME ABÉT, Budapest, Hungary) Ibolya Leveles (BME ABÉT, Hungary, Hungary)email: vertessy@mail.bme.huThe enzyme family of dUTPases are involved in preventive DNA repair by sanitizing the dNTP pool via hydrolyzing dUTP into dUMP and inorganic pyrophosphate (1). Metagenomic analyses revealed that dUTPases are present in samples isolated from extreme conditions with regard to pollutants, salt and thermal conditions (2). As to present, two major families of dUTPases are described. On the one hand, beta-sheeted subunits form a symmetric trimer wherein the three active sites are constituted by conserved sequence motifs from all the three subunits. On the other hand, alpha-helical subunits form a dimeris arrangement with active sites situated at the dimer interface.

We found that the within the trimeric dUTPase fold, several organisms encode the respective gene that contains three copies of the subunit in a linked arrangement such that the resulting protein product contains the three subunits in covalent linkage within one single polypeptide chain. The evolutionary pathway leading the the triplication of the ancient dUTPase gene necessarily involves a dimeric evolutionary intermediate (3).

Here we investigate if such evolutionary intermediate dUTPase enzyme may function as a catalytically active species, in constrast to the general arrangement of this enzyme family. We characterize the dimeric enzyme using enzyme kinetics and biophysical techniques. We also crystallize this evolutionary intermediate and provide structural insights into the organization of the active site.
References:

1: Vértessy BG, Tóth J. Keeping uracil out of DNA: physiological role, structure and catalytic mechanism of dUTPases. Acc Chem Res. 2009 Jan 20;42(1):97-106.



2: Szalkai B, Scheer I, Nagy K, Vértessy BG, Grolmusz V. The metagenomic telescope. PLoS One. 2014 Jul 23;9(7):e101605.



3: Benedek A, Horváth A, Hirmondó R, Ozohanics O, Békési A, Módos K, Révész Á, Vékey K, Nagy GN, Vértessy BG. Potential steps in the evolution of a fused trimeric all-β dUTPase involve a catalytically competent fused dimeric intermediate. FEBS J. 2016 Sep;283(18):3268-86.



Keywords: folding, homology, dUTPases