Skip navigation
   
 
Scholarly Communication
Contacts

Characterization of a Nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates

Fisher, David I; Cartwright, Jared L; Harashima, Hideyoshi; Kamiya, Hiroyuki and McLennan, Alexander G (2004) Characterization of a Nudix hydrolase from Deinococcus radiodurans with a marked specificity for (deoxy)ribonucleoside 5'-diphosphates. BMC Biochemistry, 5 . Article Number: 7. ISSN 1471-2091

[img]
Preview
PDF
Available under License Creative Commons Attribution.

322Kb

Official URL: http://www.biomedcentral.com/1471-2091/5/7

Abstract

Background: Nudix hydrolases form a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. The genome of the radioresistant bacterium Deinococcus radiodurans encodes 25 nudix hydrolases, an unexpectedly large number. These may contribute to radioresistance by removing mutagenic oxidised and otherwise damaged nucleotides. Characterisation of these hydrolases is necessary to understand the reason for their presence. Here, we report the cloning and characterisation of the DR0975 gene product, a nudix hydrolase that appears to be unique to this organism. Results: The DR0975 gene was cloned and expressed as a 20 kDa histidine-tagged recombinant product in Escherichia coli. Substrate analysis of the purified enzyme showed it to act primarily as a phosphatase with a marked preference for (deoxy)nucleoside 5'-diphosphates (dGDP > ADP > dADP > GDP > dTDP > UDP > dCDP > CDP). Km for dGDP was 110 μM and kcat was 0.18 s-1 under optimal assay conditions (pH 9.4, 7.5 mM Mg2+). 8-Hydroxy-2'-deoxyguanosine 5'- diphosphate (8-OH-dGDP) was also a substrate with a Km of 170 μM and kcat of 0.13 s-1. Thus, DR0975 showed no preference for 8-OH-dGDP over dGDP. Limited pyrophosphatase activity was also observed with NADH and some (di)adenosine polyphosphates but no other substrates. Expression of the DR0975 gene was undetectable in logarithmic phase cells but was induced at least 30-fold in stationary phase. Superoxide, but not peroxide, stress and slow, but not rapid, dehydration both caused a slight induction of the DR0975 gene. Conclusion: Nucleotide substrates for nudix hydrolases conform to the structure NDP-X, where X can be one of several moieties. Thus, a preference for (d)NDPs themselves is most unusual. The lack of preference for 8-OH-dGDP over dGDP as a substrate combined with the induction in stationary phase, but not by peroxide or superoxide, suggests that the function of DR09075 may be to assist in the recycling of nucleotides under the very different metabolic requirements of stationary phase. Thus, if DR0975 does contribute to radiation resistance, this contribution may be indirect.

Item Type:Article
Additional Information:Published: 17 May 2004. 8 pages (page numbers not for citation purposes).
Uncontrolled Keywords:intracellular nucleotides; toxic derivatives; mutagenic oxidised; DR0975 gene
Subjects:Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Departments, Research Centres and Related Units:Academic Faculties, Institutes and Research Centres > Faculty of Science > Department of Biological Sciences
DOI:10.1186/1471-2091-5-7
Publisher's Statement:© 2004 Fisher et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. This article is available from: http://www.biomedcentral.com/1471-2091/5/7
Related URLs:
Refereed:Yes
Status:Published
ID Code:781
Deposited On:07 Jul 2008 10:07
Last Modified:19 May 2011 20:20

Repository Staff Only: item control page

   
Search


Full text only
Peer reviewed only

Browse
Cross Archive Search
Find
Top 50 authors
Top 50 items
[more statistics]
 
   

These pages are maintained by Library Staff @ University of Liverpool Library

 

All pages © The University of Liverpool, 2004 | Disclaimer | Accessibility | Staff | Students