CRYSTAL STRUCTURE OF FIV dUTP PYROPHOSPHATASE. G. Sridhar Prasad, E. A. Stura, D. E. McRee, C. Hasselkus-Light, G. S. Laco, J. H. Elder, C. D. Stout, Department of Molecular Biology, The Scripps Research Institute, La Jolla, 92037

The crystal structure of the dUTP pyrophosphatase (dUTPase) from feline immunodeficiency virus (FIV) has a similar fold to the E. col i enzyme ( 1 ) in which the C-terminal strand of an anti-parallel [[beta]]-sandwich participates in the [[beta]]-sheet of an adjacent subunit to form an interdigitated, biologically functional trimer. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate. By maintaining low cellular concentrations of dUTP the enzyme prevents incorporation of uracil into DNA, which if unchecked leads to numerous strand breaks due to extensive excision repair by uracil-DNA-glycosylase. As an essential enzyme of nucleotide metabolism, dUTPase is a potential target for drug design. The objective of this study is to understand the structural basis for the substrate specificity, Mg²⁺ ion dependence and chemical mechanism of dUTPase. The active site of the E. Coli enzyme has not yet been defined ( 1).

Three crystal forms of FIV dUTPase have been obtained using recombinant protein expressed in both E. Coli and baculovirus (monomer MW 14,350) (2). The structure has been solved by MIR methods using a P6₃ crystal form containing two monomers per asymmetric (one trimer on the 6₃ screw axis and one trimer on the 3-fold axis). For two Hg derivatives the figure of merit is 0.67 to 2.7Å resolution (phasing power 2.90 and 2.97). Complete native data to 1.8Å resolution has been collected at 90deg.K. Refinement of the structure is in progress. The P6₃ crystal form requires Mg²⁺ and also binds Sm³⁺, Gd³⁺ and Yb³⁺ at a site on the 3-fold axis. However, in soaking experiments this form does not accommodate substrates. A second crystal form, which is orthorhombic and contains one trimer per asymmetric unit is grown in the presence of dUDP, an inhibitor of the enzyme. A third crystal form, in space group P6₁, contains 4 trimers per asymmetric unit. Complete data have been collected to 3.0Å resolution for this crystal form. Progress in the study of dUTPase Mg²⁺, substrate and inhibitor complexes in these crystal forms will be reported.

1. E. S. Cedergren-Zeppezauer, et al., Nature 355, 740 (1992).

2. P. C. Wagaman, et al., Virology 196, 451 (1993); and J. H. Elder, et al., in preparation.