Hydrolysis of the 5′-p-nitrophenyl ester of TMP by oligoribonucleases (ORN) from Escherichia coli, Mycobacterium smegmatis, and human

Ah Young Park, Christopher M. Elvin, Samir Hamdan, Robert J. Wood, Nancy E. Liyou, Tamarind E. Hamwood, Phil A. Jennings, Nicholas E. Dixon*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Escherichia coli oligoribonuclease (EcoORN), encoded by the orn gene, is a 3′-5′ exonuclease that degrades short single-stranded oligoribonucleotides to rNMPs in the final step of RNA degradation. The orn gene is essential in E. coli, but not in higher organisms, and close homologues are present in other genomes from the β and γ subdivisions of the Protobacteriaceae, including many pathogenic species. We report here the expression in E. coli of orn and homologues from Mycobacterium smegmatis and human, and large-scale purification of the three enzymes. All three were found to promote the hydrolysis of the 5′-p-nitrophenyl ester of TMP (pNP-TMP) with similar values of Michaelis-Menten parameters (kcat = 100-650 min-1, KM = 0.4-2.0 mM, at pH 8.00 and 25 °C, with 1 mM Mn2+). Hydrolysis by pNP-TMP by all three enzymes depended on a divalent metal ion, with Mn2+ being preferred over Mg2+ as cofactor, and was inhibited by Ni2+. The concentration dependency of Mn2+ was examined, giving KMn values of 0.2-0.6 mM. The availability of large amounts of the purified enzymes and a simple spectrophotometric assay for ORN activity should facilitate large-scale screening for new inhibitors of bacterial oligoribonucleases.

Original languageEnglish (US)
Pages (from-to)180-187
Number of pages8
JournalProtein Expression and Purification
Volume57
Issue number2
DOIs
StatePublished - Feb 1 2008

Keywords

  • 3′-5′ exonuclease
  • DEDDh
  • Oligoribonuclease
  • RNA degradation
  • Spectrophotometric assay

ASJC Scopus subject areas

  • Biotechnology

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