THE CRYSTAL-STRUCTURE OF ESCHERICHIA-COLI PURINE NUCLEOSIDE PHOSPHORYLASE - A COMPARISON WITH THE HUMAN ENZYME REVEALS A CONSERVED TOPOLOGY

Background: Purine nucleoside phosphorylase (PNP) from Escherichia col i is a hexameric enzyme that catalyzes the reversible phosphorolysis o f 6-amino and 6-oxopurine (2'-deoxy) ribonucleosides to the free base and (2'-deoxy)ribose-1-phosphate. In contrast, human and bovine PNPs a re trimeric and accept only 6-oxopurine nucleosides as substrates. The difference in the specificities of these two enzymes has been utilize d in gene therapy treatments in which certain prodrugs are cleaved by E. coli PNP but not the human enzyme, The trimeric and hexameric PNPs show no similarity in amino acid sequence, even though they catalyze t he same basic chemical reaction. Structural comparison of the active s ites of mammalian and E. coli PNPs would provide an improved basis for the design of potential prodrugs that are specific for E. coli PNP. R esults: The crystal structure of E. coli PNP at 2.0 Angstrom resolutio n shows that the overall subunit topology and active-site location wit hin the subunit are similar to those of the subunits from human PNP an d E. coli uridine phosphorylase. Nevertheless, even though the overall geometry of the E. coli PNP active site is similar to human PNP, the active-site residues and subunit interactions are strikingly different . In E. coli PNP, the purine- and ribose-binding sites are generally h ydrophobic, although a histidine residue from an adjacent subunit prob ably forms a hydrogen bond with a hydroxyl group of the sugar. The pho sphate-binding site probably consists of two mainchain nitrogen atoms and three arginine residues, In addition, the active site in hexameric PNP is much more accessible than in trimeric PNP. Conclusions: The st ructures of human and E. coli PNP define two possible classes of nucle oside phosphorylase, and help to explain the differences in specificit y and efficiency between trimeric and hexameric PNPs. This structural data may be useful in designing prodrugs that can be activated by E. c oli PNP but not the human enzyme.