Molecular cloning and expression of a purine specific N-ribohydrolase from trypanosoma bruce1 brvcel

N-Ribohydrolases, including the inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-NH), are involved in the nucleoside salvage pathway of African trypanosomes. The iagnh gene contains an open reading frame (ORF) of 981 bp, is present as one copy/haploid genome, and is located on the size-polymorphic chromosome III or IV in the genome of '/'. h. hrncei On Southern analysis the IAG-NH probe hybridizes strongly with /'. conglense and /'. vivax and to a lesser extent with T. cruzi and l.inshmania thmovani genomic DNA. The iagnh gene is expressed in all the life-cycle-stages of T. b. brucei. There is no known significant amino acid identity of the IAG-NH sequence outside bacteria, yeast, or parasitic organisms, with only low amino acid homology occuring with the inosineuridine preferring (IU-) NH isozyme from (". fascicnlata. The iagnh (ORF) was cloned into K. coli BL21(DE3) and a soluble recombinant IAG-NH expressed and purified to > 97% homogeneity. The molecular weight of the recombinant IAG-NH, based on the amino acid sequence and observed mass, was 35.735 and 35,737, respectively. Conserved motifs DxDxxxDDxxA and AExNI have been identified in N-ribohydrolases and are associated with Ca and ribose binding.