Escherichia coli uridine phosphorylase (UP) is encoded by the udp gene and catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Only few key residues involved in UP catalysis, identified by site-directed mutagenesis and selective chemical modification studies, were reported. A recent paper reporting the crystal structure at 2.0 Angstrom resolution indicated that UP shares a high structural homology with E. coli purine nucleoside phosphorylase. This latter enzyme is better known and a number of residues in its active site have been identified. In this work, we used pentapeptide scanning mutagenesis to cause random insertions of a 5 amino acid cassette in the UP polypeptide chain. Several insertional mutants located in different regions of UP maintained or increased the enzymatic activity and provided new insights into protein structure-function relationships. Moreover, this mutagenesis approach appears to be useful for the rapid preparation of mutants that present altered enzymatic activities. (C) 2004 Elsevier Inc. All rights reserved.