Protozoa depend on purine salvage for nucleic acid synthesis. An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase). The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C. fasciculata. The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity. The open reading frame encodes 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 amu 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 purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively. The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins. A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence. Open reading frames near 1 and 47 min on the E. coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity. Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation. IU-nucleoside hydrolase from C. fasciculata and the protein expressed in E. coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively. Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees. Two subunits of the tetrameric enzyme are present in the asymmetric unit. The following paper reports the X-ray crystal structure for this enzyme.

• PMID: 8634237
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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Gopaul DN, Meyer SL, Degano M, Sacchettini JC, Schramm VL

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