Characterization of Escherichia coli Uridine Phosphorylase by Single-Site Mutagenesis

Abstract

The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. The X-ray structure of E. coli UP resolved by two different groups produced conflicting results. In order to cast some light on the E. coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro. Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity. These three residues were suggested to be involved in the nucleoside binding site. However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity. Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site. Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position. Glu198 was mutated to either Gly, Asp and Gln. All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions. Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions. Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20%. Both amino acids were suggested to take part in subunit interactions. Our results confirm the structural similarity between E. coli UP and E. coli purine nucleoside phosphorylase (PNP).

Improved synthesis of 2′-deoxyadenosine and 5-methyluridine by Escherichia coli using an auto-induction system

“…The use of whole cells to synthesis nucleoside analogues has been used increasingly (Rocchietti et al 2004;End and Schoning 2004;Tonon et al 2004), as the isolation and purification of enzymes can be avoided (Oliva et al 2004a;b;Panova et al 2007). More recently, the construction of genetically modified bacteria that express high levels of recombinant NPs have been used successfully in the production of isolated enzymes and as whole cell biocatalysts.…”

Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

“…The crystal structure of E. coli UP in a complex with substrates was analyzed (see [39–40] and the works cited therein) and the role of some amino acid residues of the catalytic site was characterized by the single-site mutagenesis [41]. It was suggested that the uracil binding site includes Gln166, the carboxamide group of which forms two strong hydrogen bonds 3N(H)···(O=)C(R)-NH 2 ···(O=)C-2.…”