Multidimensional NMR Identifies the Conformational Shift Essential for Catalytic Competence in the 60-kDa Drosophila melanogaster dUTPase Trimer

Abstract

The catalytic mechanism of dUTP pyrophosphatase (dUTPase), responsible for the prevention of uracil incorporation into DNA, involves ordering of the flexible C terminus of the enzyme. This conformational shift is investigated by multidimensional NMR on the Drosophila enzyme. Flexible segments of the homotrimer give rise to sharp resonances in the (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, which are clearly distinguishable from the background resonances of the well folded protein globule. Binding of the product dUMP or the analogues dUDP and alpha,beta-imino-dUTP to the enzyme induces a conformational change reflected in the disappearance of eight sharp resonances. This phenomenon is interpreted as nucleotide binding-induced ordering of some residues upon the folded protein globule. Three-dimensional (15)N-edited (1)H-(15)N HSQC total correlation spectroscopy (TOCSY) and (1)H-(15)N HSQC nuclear Overhauser effect spectroscopy measurements allowed clear assignment of these eight specific resonance peaks. The residues identified correspond to the conserved C-terminal sequence motif, indicating that (i) this conformational shift is amenable to NMR studies in solution even in the large trimeric molecule and (ii) formation of the closed enzyme conformer in the case of the Drosophila enzyme does not require the complete triphosphate chain of the substrate. NMR titration of the enzyme with the nucleotide ligands as well as kinetic data indicated significant deviation from the model of independent active sites within the homotrimer. The results suggest allosterism in the eukaryotic dUTPase.

Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

“…In contrast with DUT1, we found that the purified E. coli dUTPase exhibited a very low co-operativity ( h 1.3±0.4) in the saturation experiments with dUTP (results not shown). Previous NMR experiments with the Drosophila dUTPase revealed a co-operative behaviour and ligand-induced co-operative conformational changes in this eukaryotic dUTPase too [41]. Our present results support the suggestion that eukaryotic dUTPases have acquired positive co-operativity during evolution [41].…”

“…Previous NMR experiments with the Drosophila dUTPase revealed a co-operative behaviour and ligand-induced co-operative conformational changes in this eukaryotic dUTPase too [41]. Our present results support the suggestion that eukaryotic dUTPases have acquired positive co-operativity during evolution [41]. In the presence of saturating Mg 2+ concentrations (0.5 mM), DUT1 exhibited higher activity and affinity for dUTP resulting in 20 times higher catalytic efficiency compared with dITP (Table 2).…”

A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases

“…The increased polarity of the central threefold channel, which is the main structural difference between bacterial and eukaryotic dUTPases, is suggested to render the latter protein more exposed to environmental effects. This increased sensitivity to the environment is reflected not only in decreased stability, but also in ligand induced conformational changes [15], as well as in cooperativity of nucleotide binding [6,7]. It seems that increased sensitivity towards cognate ligands, presumably required in developed organisms, is coupled to some loss of protein stability.…”

The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

“…Both constructs were expressed in Escherichia coli BL21 (DE3) Rosetta cells under similar conditions (cf. also [17,31,32]).…”