Structural and Functional Insights into DR2231 Protein, the MazG-like Nucleoside Triphosphate Pyrophosphohydrolase from Deinococcus radiodurans

Abstract

Deinococcus radiodurans is among the very few bacterial species extremely resistant to ionizing radiation, UV light, oxidizing agents, and cycles of prolonged desiccation. The proteome of D. radiodurans reflects the evolutionary pressure exerted by chronic exposure to (nonradioactive) forms of DNA and protein damage. A clear example of this adaptation is the overrepresentation of protein families involved in the removal of non-canonical nucleoside triphosphates (NTPs) whose incorporation into nascent DNA would promote mutagenesis and DNA damage. The three-dimensional structure of the DR2231 protein has been solved at 1.80 Å resolution. This protein had been classified as an all-␣-helical MazG-like protein. The present study confirms that it holds the basic structural module characteristic of the MazG superfamily; two helices form a rigid domain, and two helices form a mobile domain and connecting loops. Contrary to what is known of MazG proteins, DR2231 protein shows a functional affinity with dUTPases. Enzymatic and isothermal calorimetry assays have demonstrated high specificity toward dUTP but an inability to hydrolyze dTTP, a typical feature of dUTPases. Co-crystallization with the product of hydrolysis, dUMP, in the presence of magnesium or manganese cations, suggests similarities with the dUTP/dUDP hydrolysis mechanism reported for dimeric dUTPases. The genome of D. radiodurans encodes for all enzymes required for dTTP synthesis from dCMP, thus bypassing the need of a dUTPase. We postulate that DR2231 protein is not essential to D. radiodurans and rather performs "house-cleaning" functions within the framework of oxidative stress response. We further propose DR2231 protein as an evolutionary precursor of dimeric dUTPases.

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

“…In the crystal structure (PDB code: 1EXC) for the Maf enzymes, the ligand dUTP is bound in a catalytically incompetent conformation within the active site: only the triphosphate part of the nucleotide is recognized by the enzyme; other parts are not bound [116]. [100,102] sequence conservation. Although human and EpsteinBarr viral dUTPases show only 19% identity [30,39,40], the same fold is displayed by the protein.…”

“…The interest in these enzymes is highly motivated on the one hand by the peculiar active site architecture and, on the other hand, by the possibility of using these dUTPases as targets for developing drugs against neoplastic, as well as infectious diseases [31,[56][57][58][59][60][61][62][63][64][65] [100]. All-b dUTPases also include monomeric dUTPases [40].…”

“…Subsequently, in addition to the hydrolysis of canonical (d)NTPs, a rather diverse substrate specificity on noncanonical nucleotides was reported by the characterization of various MazG homologues. The biological role of tandem MazG domain proteins was reported to be the regulation of ppGpp (guanosine 3 0 ,5 0 -bispyrophosphate) cellular levels, extending the period of bacterial cell survival under nutritional stress [99,100]. MazG from Deinococcus radiodurans displayed not only structural, but also functional similarity to dimeric dUTPases by its high specificity to dUTP along with the inability to hydrolyze dTTP, leading to the hypothesis of a MazGlike ancestor as the evolutionary precursor of dimeric dUTPases [100].…”

“…The biological role of tandem MazG domain proteins was reported to be the regulation of ppGpp (guanosine 3 0 ,5 0 -bispyrophosphate) cellular levels, extending the period of bacterial cell survival under nutritional stress [99,100]. MazG from Deinococcus radiodurans displayed not only structural, but also functional similarity to dimeric dUTPases by its high specificity to dUTP along with the inability to hydrolyze dTTP, leading to the hypothesis of a MazGlike ancestor as the evolutionary precursor of dimeric dUTPases [100]. The RS21-C6 mammalian MazG displayed preference for 5-halo-dCTP analogues [101], whereas the MazG from Mycobacteria was shown to be responsible for the degradation of 5-OH-dCTP [99].…”

“…The RS21-C6 mammalian MazG displayed preference for 5-halo-dCTP analogues [101], whereas the MazG from Mycobacteria was shown to be responsible for the degradation of 5-OH-dCTP [99]. Numerous structural and mutational studies have shed light on the active site architecture and mechanism of these enzymes [100,102,103]. In the MazG enzymes, the EEXXE 12-28 EXXD conserved sequence motif positions Mg 2+ in the active site, identically to other enzyme representatives from the all-a NTPases superfamily (Fig.…”