Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli |
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Authors: | Brock Arivett Mary Farone Ranjith Masiragani Andrew Burden Shelby Judge Adedoyin Osinloye Claudia Minici Massimo Degano Matthew Robinson Paul Kline |
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Institution: | 1. Department of Chemistry, Middle Tennessee State University, Murfreesboro, TN 37132, USA;2. Department of Biology, Middle Tennessee State University, Murfreesboro, TN 37132, USA;3. Division of Immunology, Transplantation and Infectious Diseases, Scientific Institute San Raffaele, Milan Italy |
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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. |
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Keywords: | BICINE N N-bis(2-hydroxyethyl)glycine BSA bovine serum albumin CHES 2-(cyclohexylamino)ethanesulfonic acid CU-NH cytidine-uridine nucleoside hydrolase HEPES N-(2-hydroxyethyl)piperazine-N&prime -4-butanesulfonic acid) IAG-NH inosine&ndash adenosine&ndash guanosine nucleoside hydrolase IG-NH inosine&ndash guanosine nucleoside hydrolase IPTG isopropyl-β-D-1-thiogalactopyranoside IU-NH inosine&ndash uridine nucleoside hydrolase LB Luria Broth MES 2-(N-morpholino)ethanesulfonic acid PIPES 1 4-piperazinediethanesulfonic acid |
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