Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy)ribonucleoside 5'-Di- and triphosphates

Abstract

The genome of the extremely radiation resistant bacterium Deinococcus radiodurans encodes 21 Nudix hydrolases, of which only two have been characterized in detail. Here we report the activity and crystal structure for DR_0079, the first Nudix hydrolase observed to have a marked preference for cytosine ribonucleoside 5'-diphosphate (CDP) and cytosine ribonucleoside 5'-triphosphate (CTP). After CDP and CTP, the next most preferred substrates for DR_0079, with a relative activity of <50%, were the corresponding deoxyribose nucleotides, dCDP and dCTP. Hydrolase activity at the site of the phosphodiester bond was corroborated using (31)P NMR spectroscopy to follow the phosphorus resonances for three substrates, CDP, IDP, and CTP, and their hydrolysis products, CMP + P(i), IMP + P(i), and CMP + PP(i), respectively. Nucleophilic substitution at the beta-phosphorus of CDP and CTP was established, using (31)P NMR spectroscopy, by the appearance of an upfield-shifted P(i) resonance and line-broadened PP(i) resonance, respectively, when the hydrolysis was performed in 40% H(2)(18)O-enriched water. The optimal activity for CDP was at pH 9.0-9.5 with the reaction requiring divalent metal cation (Mg(2+) > Mn(2+) > Co(2+)). The biochemical data are discussed with reference to the crystal structure for DR_0079 that was determined in the metal-free form at 1.9 A resolution. The protein contains nine beta-strands, three alpha-helices, and two 3(10)-helices organized into three subdomains: an N-terminal beta-sheet, a central Nudix core, and a C-terminal helix-turn-helix motif. As observed for all known structures of Nudix hydrolases, the alpha-helix of the "Nudix box" is one of two helices that sandwich a "four-strand" mixed beta-sheet. To identify residues potentially involved in metal and substrate binding, NMR chemical shift mapping experiments were performed on (15)N-labeled DR_0079 with the paramagnetic divalent cation Co(2+) and the nonhydrolyzable substrate thymidine 5'-O-(alpha,beta-methylenediphosphate) and the results mapped onto the crystal structure.

Fig. 1

(A) Molscript ribbon representation of the crystal structure of DR_0079 in the metal-free form (PDB ID = 2O5F). The β-strands are colored blue, α-helices red, and 3₁₀-helices magenta. Residues 1 – 34 = N-terminal anti-parallel β-sheet; residues 35–140 = Nudix core; residues 141– 171 = C-terminal helix-turn-helix motif. (B) Secondary structure diagram of DR_0079. The α-helices are drawn as red ovals and 3₁₀-helices as magenta ovals with the residue number of the beginning and the end of each element shown. The β-strands are drawn as solid blue arrows with each residue indicated within a box. The β-sheets contain bulges at residues G20 and L102. A solid pink line between β-sheet residues indicates dual hydrogen bonds between two residues in an anti-parallel β-sheet.

Fig. 1

(A) Molscript ribbon representation of the crystal structure of DR_0079 in the metal-free form (PDB ID = 2O5F). The β-strands are colored blue, α-helices red, and 3₁₀-helices magenta. Residues 1 – 34 = N-terminal anti-parallel β-sheet; residues 35–140 = Nudix core; residues 141– 171 = C-terminal helix-turn-helix motif. (B) Secondary structure diagram of DR_0079. The α-helices are drawn as red ovals and 3₁₀-helices as magenta ovals with the residue number of the beginning and the end of each element shown. The β-strands are drawn as solid blue arrows with each residue indicated within a box. The β-sheets contain bulges at residues G20 and L102. A solid pink line between β-sheet residues indicates dual hydrogen bonds between two residues in an anti-parallel β-sheet.

Fig. 2

(A) Expansion of the loop-helix-loop Nudix box with the side chains of the nine highly conserved residues highlighted, labeled, and colored according to atom type. The regions that immediately surround the Nudix box are highlighted on the cartoon (B) and surface (C) structure of DR_0079. Red = Nudix box, purple = N-terminal β-sheet, blue = loop r1, cyan = loop r3, magenta = rest of β5, yellow = β3, and green = C-terminal helix-turn-helix.

Fig. 3

Molscript ribbon representation of two molecules of DR_0079 in the asymmetric unit highlighting the intermolecular interface. The β-strands are colored blue, the alpha- and 3₁₀-helices red, and the turns and loops grey.

Fig. 4

A) Relative substrate activity of DR_0079 towards a variety of nucleoside di- and triphosphate substrates. B) Nudix hydrolase activity of DR_0079 as a function of metal cation. The divalent cation requirements (metal profile) were determined towards CDP in the presence of 5 mM Mg²⁺ or 0.5 mM other divalent cation (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Ca²⁺, or Cu²⁺).

Fig. 5

One-dimensional ³¹P spectra of CDP, CTP, and IDP alone and at various time intervals after the addition of DR_0079 to the NMR tube. (A) CDP alone, (B) CDP + DR_0079 immediately after protein addition with the inset showing an expansion (0.4 ppm) of the Pi resonance in the presence of 40% H₂¹⁸O, (C) IDP alone, (D) CDP + DR_0079 after 50 min. (E) CTP alone, (F) CTP + DR_0079 at the completion of the reaction (~12 hours later) with the inset showing an expansion (0.5 ppm) of the downfield region. All spectra were recorded at a ¹H resonance frequency of 600 MHz, 37°C, in buffer containing 50 mM Tris, 10 mM MgCl₂, pH 9.1.

Fig. 6

A) Overlay of the ¹H–¹⁵N HSQC spectra of DR_0079 in the absence (red) and presence (blue) of ~1:1 molar ratio of CoCl₂. The chemical shifts of backbone amide and identified side chain amine resonances that disappear or shift (underlined) upon the addition of CoCl₂ are labeled. Spectra were collected at a ¹H resonance frequency of 900 MHz, 25°C, in buffer containing 100 mM KCl, 20 mM potassium phosphate, 2 mM DTT, pH 7.1. B + C) Resonances for residues that disappear (marine blue) or move (slate) upon the addition of CoCl₂ to DR_0079 are mapped onto the structure (2O5F) and shown in two orientations that differ by ~180°. The metal cation likely rests over G70 (magenta), the residue with the largest chemical shift perturbation with the addition of MgCl₂ (27). Next to it, colored orange, is E85, the nearest glutamic acid side chain (the ¹H^N resonance of E85 overlaps with A160 and it is not possible to determine if this residue disappears with the addition of CoCl₂). In the cleft there is a ring of lysine and arginine residues and these are colored red (R24, R53, K57, and K156). Directly behind the ring of positively charged residues is an aromatic tryptophan side chain (W31) that is colored yellow in orientation A. This tryptophan’s location in the cleft is more obvious in orientation B.

Fig. 7

A) Overlay of the ¹H–¹⁵N HSQC spectra of DR_0079 in the absence (black) and presence (red) of ~4:1 molar ratio of TMP-CP:DR_0079. The chemical shifts of backbone amide resonances that disappear or shift (underlined) upon the addition of TMP-CP are labeled. Spectra were collected at a ¹H resonance frequency of 600 MHz, 25°C, in buffer containing 100 mM KCl, 100 mM MgCl₂, 20 mM potassium phosphate, 2 mM DTT, pH 7.1. B + C) Residues of backbone amides that disappear (dark blue) or shift slightly (light blue) upon the addition of TMP-CP to DR_0079 at a substrate:protein molar ratio of ~4.0:1.0 are mapped unto the structure of DR_0079 and shown in two orientations that differ by ~180°. Also highlighted are E85 and E89 (orange), the residues that most likely bind the divalent cation, and G70 (magenta), the residue the cation may sit over.