INOSINE-URIDINE NUCLEOSIDE HYDROLASE FROM CRITHIDIA-FASCICULATA - GENETIC-CHARACTERIZATION, CRYSTALLIZATION, AND IDENTIFICATION OF HISTIDINE-241 AS A CATALYTIC SITE RESIDUE

Protozoa depend on purine salvage for nucleic acid synthesis. An abund ant salvage enzyme in Crithidia fasciculata is the inosine-uridine nuc leoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to th e amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli an d the protein purified to >99% homogeneity. The open reading frame enc odes a protein of 315 amino acids. Enzyme purified from C. fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 a mu by mass spectrometry. Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196. A pET3d-IUNH construct expressed in E . coli introduced MetAla instead of MetPro at the N-terminus. Enzyme p urified from this construct also had a processed N-terminus and gave p redicted and observed masses of 34 168 and 34 170 amu, respectively. T he amino acid sequence for IU-nucleoside hydrolase has no close relati ves among the known proteins. A cDNA clone of unknown function from Le ishmania major shows near identity in the N-terminal deduced amino aci d sequence. Open reading frames near 1 and 47 min on the E. coli chrom osome and from two yeast genomes encode for proteins of similar size w ith substantial amino acid identity. Mutation of His241Ala caused a 21 00-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) w ith p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leavin g group activation. IU-nucleoside hydrolase from C. fasciculata and th e protein expressed in E. coli were crystallized and diffract to 2.5 a nd 2.1 Angstrom resolution, respectively. Both belong to the P2(1)2(1) 2 orthorhombic space group with unit cell parameters a = 63.5 Angstrom , b = 131.9 Angstrom, c = 90.1 Angstrom, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asy mmetric unit. The following paper reports the X-ray crystal structure for this enzyme.