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 mu M. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocydic 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.(C) 2014 Elsevier B.V. All rights reserved.
Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli / Arivett, Brock; Farone, Mary; Masiragani, Ranjith; Burden, Andrew; Judge, Shelby; Osinloye, Adedoyin; Minici, Claudia; Degano, Massimo; Robinson, Matthew; Kline, Paul. - In: BIOCHIMICA ET BIOPHYSICA ACTA. - ISSN 0006-3002. - 1844:3(2014), pp. 656-662. [10.1016/j.bbapap.2014.01.010]
Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli
Degano, MassimoFormal Analysis
;
2014-01-01
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 mu M. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocydic 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.(C) 2014 Elsevier B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
