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J. Biol. Chem., Vol. 279, Issue 17, 17932-17944, April 23, 2004

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Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

KINETIC CHARACTERIZATION, FOLDING, AND CRYSTALLOGRAPHIC STUDIES OF THE HOMOTRIMERIC DROSOPHILA ENZYME^*

Júlia Kovári, Orsolya Barabás, Enikõ Takács, Angéla Békési, Zsófia Dubrovay, Veronika Pongrácz, Imre Zagyva, Timea Imre¶, Pál Szabó¶, and Beáta G. Vértessy||

From the Institute of Enzymology, Biological Research Center (BRC), Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary, Loránd Eötvös University, Department of Theoretical Chemistry, POB 32, H-1518 Budapest, Hungary, and the ^¶Department of Mass Spectrometry, Chemical Research Center, Hungarian Academy of Sciences, Hungarian Academy of Science, POB 17, H-1525, Budapest, Hungary

dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54 °C, 23 °C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg²⁺ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

Received for publication, December 12, 2003 , and in revised form, January 13, 2004.

^* This work was supported by Országos Tudományos Kutatási Alap Grants T034120, TS044730, and M27852 (to B. G. V.) and F025602 (to P. S.), and T034944 and T043538 from the Hungarian National Research Foundation; Grant 55000342 from the Howard Hughes Medical Institute (to B. G. V.); a grant from the Alexander von Humboldt Foundation; a grant from the Aventis/Institut de France Scientia Europeae Prize (to B. G. V.); and Grant QLK2-CT-2002-90436 from the European Union. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Institute of Enzymology, BRC, Hung. Acad. Sci., POB 7, H-1518 Budapest, Hungary. Tel.: 361-279-3100; Fax: 361-466-5465; E-mail: vertessy{at}enzim.hu.

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