Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli

Brock Arivett, Mary Farone, Ranjith Masiragani, Andrew Burden, Shelby Judge, Adedoyin Osinloye, Claudia Minici, Massimo Degano, Matthew Robinson, Paul Kline

Research output: Contribution to journalArticlepeer-review

Abstract

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.

Original languageEnglish
Pages (from-to)656-662
Number of pages7
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1844
Issue number3
DOIs
Publication statusPublished - Mar 2014

Keywords

  • Escherichia coli
  • Nucleoside hydrolase
  • RihC

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

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