Effect of an Asp80Ala substitution on the binding of dUTP and dUMP to Trypanosoma cruzi dUTPase.

dUTPase (deoxyuridine 5'-triphosphate nucleotide hydrolase) is an enzyme responsible for maintaining low levels of intracellular dUTP and thus prevents uracil incorporation into DNA by DNA polymerases during replication and repair processes. The thermodynamics of binding for both dUTP and dUMP (deoxyuridine 5'-monophosphate) to the D80A mutant form of Trypanosoma cruzi dUTPase have been investigated by fluorescence spectroscopy and high-sensitivity isothermal titration calorimetry. In the presence of magnesium, approximately a 30-fold decrease in the value of the k(cat) and a 15-fold increase in the K(m) for dUTP hydrolysis was calculated while a 5-fold decrease was observed in the affinity for dUMP. In the absence of magnesium, the affinity for dUTP binding was similar for both enzymes while that for dUMP was lowered 3-fold as a consequence of the mutation. Calorimetric titrations in several buffers with different ionization heats rendered similar proton exchanges during the binding of dUMP. Thus, apparently the side chain of Asp 80 does not seem to vary its protonation state during the binding process. The enthalpy change values for the D80A mutant hardly change with temperature and, in addition, were Mg(2+) independent. We conclude that the D80A mutation induces only a slight conformational change in the active site yet results in a significant alteration of nucleotide binding and modifies the ability of the enzyme to discriminate between dUTP and dUMP when magnesium is present.

The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding fold.

dUTPase is an essential enzyme involved with nucleotide metabolism and replication. We report here the X-ray structure of Trypanosoma cruzi dUTPase in its native conformation and as a complex with dUDP. These reveal a novel protein fold that displays no structural similarities to previously described dUTPases. The molecular unit is a dimer with two active sites. Nucleotide binding promotes extensive structural rearrangements, secondary structure remodeling, and rigid body displacements of 20 A or more, which effectively bury the substrate within the enzyme core for the purpose of hydrolysis. The molecular complex is a trapped enzyme-substrate arrangement which clearly demonstrates structure-induced specificity and catalytic potential. This enzyme is a novel dUTPase and therefore a potential drug target in the treatment of Chagas' disease.

Characterization of deoxyuridine 5'-triphosphate nucleotidohydrolase from Trypanosoma cruzi.

We report the cloning and kinetic characterization of Trypanosoma cruzi deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) whose coding sequence was isolated by genetic complementation in Escherichia coli. The deduced amino acid sequence was similar to Leishmania major dUTPase although it exhibits an amino acid insertion which is sensitive to protease inactivation. The catalytically active species of the enzyme is a dimer and a detailed kinetic characterization showed that it is highly specific for dUTP and dUDP. The general observation that dUTPases from the Trypanosomatidae differ in sequence, conformation and substrate specificity suggests that a different family of dUTPases exists in certain organisms, which may be exploited as drug targets against infectious diseases.

Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1.

E2 enzymes catalyze attachment of ubiquitin and ubiquitin-like proteins to lysine residues directly or through E3-mediated reactions. The small ubiquitin-like modifier SUMO regulates nuclear transport, stress response, and signal transduction in eukaryotes and is essential for cell-cycle progression in yeast. In contrast to most ubiquitin conjugation, the SUMO E2 enzyme Ubc9 is sufficient for substrate recognition and lysine modification of known SUMO targets. Crystallographic analysis of a complex between mammalian Ubc9 and a C-terminal domain of RanGAP1 at 2.5 A reveals structural determinants for recognition of consensus SUMO modification sequences found within SUMO-conjugated proteins. Structure-based mutagenesis and biochemical analysis of Ubc9 and RanGAP1 reveal distinct motifs required for substrate binding and SUMO modification of p53, IkappaBalpha, and RanGAP1.