TY - JOUR
T1 - Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli
AU - Arivett, Brock
AU - Farone, Mary
AU - Masiragani, Ranjith
AU - Burden, Andrew
AU - Judge, Shelby
AU - Osinloye, Adedoyin
AU - Minici, Claudia
AU - Degano, Massimo
AU - Robinson, Matthew
AU - Kline, Paul
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - Escherichia coli
KW - Nucleoside hydrolase
KW - RihC
UR - http://www.scopus.com/inward/record.url?scp=84894148833&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84894148833&partnerID=8YFLogxK
U2 - 10.1016/j.bbapap.2014.01.010
DO - 10.1016/j.bbapap.2014.01.010
M3 - Article
C2 - 24473221
AN - SCOPUS:84894148833
VL - 1844
SP - 656
EP - 662
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
SN - 1570-9639
IS - 3
ER -