Discovery and substrate specificity of a novel nucleoside hydrolase

Abstract

Modified nucleotides are present in various RNA species and are critical for folding, stability, and interaction with cellular proteins. Biosynthesis of pyrimidine nucleotides and their modified derivatives in RNA is well studied, yet much less is known about the cellular degradation of these compounds and their salvage. Using methods of bacterial genetics, an E. coli strain, unable to convert 20-O-methyluridine into uracil and therefore to grow on minimal medium, was created. The growth phenotype of such mutant was restored by introducing a genome fragment from the metagenomic libraries. Such fragment contains 2 ORFs, one encoding a bacterial nucleoside hydrolase, and the other –a ribose aldolase. The recombinant nucleoside hydrolase was expressed in E. coli, purified, and its substrate specificity was investigated.