3-DIMENSIONAL STRUCTURE OF THE INOSINE-URIDINE NUCLEOSIDE N-RIBOHYDROLASE FROM CRITHIDIA-FASCICULATA

Protozoan parasites rely on the host for purines since they lack a ne novo synthetic pathway. Crithidia fasciculata salvages exogenous inosi ne primarily through hydrolysis of the N-ribosidic bond using several nucleoside hydrolases. The most abundant nucleoside hydrolase is relat ively nonspecific but prefers inosine and uridine as substrates. Here we report the three-dimensional structure of the inosine-uridine nucle oside hydrolase (IU-NH) from C. fasciculata determined by X-ray crysta llography at a nominal resolution of 2.5 Angstrom. The enzyme has an o pen (alpha,beta) structure which differs from the classical dinucleoti de binding fold. IU-nucleoside hydrolase is composed of a mixed eight- stranded beta sheet surrounded by six alpha helices and a small C-term inal lobe composed of four alpha helices. Two short antiparallel beta strands are involved in intermolecular contacts. The catalytic pocket is located at the C-terminal end of beta strands beta 1 and beta 4. Fo ur aspartate residues are located at the bottom of the cavity in a geo metry which suggests interaction with the ribose moiety of the nucleos ide. These groups could provide the catalytically important interactio ns to the ribosyl hydroxyls and the stabilizing anion for the oxycarbo nium-like transition state. Histidine 241, located on the side of the active site cavity, is the proposed proton donor which facilitates pur ine base departure [Gopaul, D. N., Meyer, S. L., Degano, M., Sacchetti ni, J. C., & Schramm, V. L. (1996) Biochemistry 35, 5963-5970]. The su bstrate binding site is unlike that from purine nucleoside phosphoryla se, phosphoribosyltransferases, or uracil DNA glycosylase and thus rep resents a novel architecture for general acid-base catalysis. This det ailed knowledge of the architecture of the active site, together with the previous transition state analysis [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-1079 5], allows analysis of the interactions leading to catalysis and an ex planation for the tight-binding inhibitors of the enzyme [Schramm, V. L., Horenstein, B. A., & Kline, P. C. (1994) J. Biol. Chem. 269, 18259 -18262].