Characterization of inosine-uridine nucleoside hydrolase (RihC) from Escherichia coli.
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 480μM. 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.
Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Brock
B
Arivett
Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Mary
M
Farone
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Ranjith
R
Masiragani
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Andrew
A
Burden
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Shelby
S
Judge
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Adedoyin
A
Osinloye
Division of Immunology, Transplantation and Infectious Diseases, Scientific Institute San Raffaele, Milan Italy.
Claudia
C
Minici
Division of Immunology, Transplantation and Infectious Diseases, Scientific Institute San Raffaele, Milan Italy.
Massimo
M
Degano
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA.
Matthew
M
Robinson
Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA. Electronic address: paul.kline@mtsu.edu.
Paul
P
Kline
D002384:Catalysis; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
D012997:Solvents; D009699:N-Glycosyl Hydrolases; C096466:inosine-uridine hydrolase
Escherichia coli; Nucleoside hydrolase; RihC