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

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 microM 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 microM 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.

Figure 1

Lineweaver-Burk plot of the hydrolysis of dGDP and 8-OH-dGDP by DR0975 protein. Assays were performed by HPLC as described in Materials and Methods.

Figure 2

Expression analysis of the DR0975 gene in cultures exposed todifferent conditions. The level of DR0975 mRNA expressed in logarithmic (log) and stationary (sta) phase cells, in cells exposed to hydrogen peroxide (per) or menadione (sup) and from cells subjected to rapid desiccation by freeze drying (rap) or slow (slo) dehydration, followed by rehydration in each case, was quantified by RT-PCR as described in Materials and Methods. Each value represents the mean of triplicate amplifications of each of three independently prepared RNA samples for each condition. The amount of amplified product was determined by densitometry by comparison with the concentrations of the standards amplified from genomic DNA by gene-specific primers. The limit of detection was 4.23 × 10^-16 M.