Deinococcus radiodurans DR2231 is a two-metal-ion mechanism hydrolase with exclusive activity on dUTP.

DR2231 from Deinococcus radiodurans was previously functionally and structurally characterized as an all-α NTP pyrophosphohydrolase with specific dUTPase activity. dUTPases have a central role in the regulation of dUTP intracellular levels and dTTP nucleotide metabolism. DR2231 presents a conserved dimetal catalytic site, similar to all-α dimeric dUTPases, but contrary to these enzymes, it is unable to process dUDP. In this article, we present functional and structural evidence of single-point mutations that affect directly or indirectly the enzyme catalysis and provide a complete description of the all-α NTP pyrophosphohydrolase mechanism. Activity assays, isothermal titration calorimetry and the crystal structures of these mutants obtained in complex with dUMP or a dUTP analogue aid in probing the reaction mechanism. Our results demonstrate that the two metals are necessary for enzyme processing and also important to modulate the substrate binding affinity. Single-point mutations located in a structurally mobile lid-like loop show that the interactions with the nucleoside monophosphate are essential for induction of the closed conformation and ultimately for substrate processing. ß- and γ-phosphates are held in place through coordination with the second metal, which is responsible for the substrate 'gauche' orientation in the catalytic position. The lack of sufficient contacts to orient the dUDP ß-phosphate for hydrolysis explains DR2231 preference towards dUTP. Sequence and structural similarities with MazG proteins suggest that a similar mechanism might be conserved within the protein family. DATABASE: Structural data are available in the PDB under the accession numbers 5HVA, 5HWU, 5HX1, 5HYL, 5I0J, 5HZZ, 5I0M.

The crystal structure of Pseudomonas putida azoreductase - the active site revisited.

The enzymatic degradation of azo dyes begins with the reduction of the azo bond. In this article, we report the crystal structures of the native azoreductase from Pseudomonas putida MET94 (PpAzoR) (1.60 Å), of PpAzoR in complex with anthraquinone-2-sulfonate (1.50 Å), and of PpAzoR in complex with Reactive Black 5 dye (1.90 Å). These structures reveal the residues and subtle changes that accompany substrate binding and release. Such changes highlight the fine control of access to the catalytic site that is required by the ping-pong mechanism, and in turn the specificity offered by the enzyme towards different substrates. The topology surrounding the active site shows novel features of substrate recognition and binding that help to explain and differentiate the substrate specificity observed among different bacterial azoreductases.

Endo-ß-D-1,4-mannanase from Chrysonilia sitophila displays a novel loop arrangement for substrate selectivity.

The crystal structure of wild-type endo-ß-D-1,4-mannanase (EC 3.2.1.78) from the ascomycete Chrysonilia sitophila (CsMan5) has been solved at 1.40 Å resolution. The enzyme isolated directly from the source shows mixed activity as both an endo-glucanase and an endo-mannanase. CsMan5 adopts the (ß/α)(8)-barrel fold that is well conserved within the GH5 family and has highest sequence and structural homology to the GH5 endo-mannanases. Superimposition with proteins of this family shows a unique structural arrangement of three surface loops of CsMan5 that stretch over the active centre, promoting an altered topography of the binding cleft. The most relevant feature results from the repositioning of a long loop at the extremity of the binding cleft, resulting in a shortened glycone-binding region with two subsites. The other two extended loops flanking the binding groove produce a narrower cleft compared with the wide architecture observed in GH5 homologues. Two aglycone subsites (+1 and +2) are identified and a nonconserved tryptophan (Trp271) at the +1 subsite may offer steric hindrance. Taken together, these findings suggest that the discrimination of mannan substrates is achieved through modified loop length and structure.

Structural and functional insights into DR2231 protein, the MazG-like nucleoside triphosphate pyrophosphohydrolase from Deinococcus radiodurans.

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-α-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.