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Deinococcus radiodurans DR 2231 is a two‐metal‐ion mechanism hydrolase with exclusive activity on d UTP
Author(s) -
Mota Cristiano S.,
Gonçalves Ana Maria D.,
Sanctis Daniele
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13923
Subject(s) - deinococcus radiodurans , isothermal titration calorimetry , hydrolase , biochemistry , chemistry , active site , nucleotide , enzyme , allosteric regulation , protein data bank (rcsb pdb) , stereochemistry , dna , gene
DR 2231 from Deinococcus radiodurans was previously functionally and structurally characterized as an all‐α NTP pyrophosphohydrolase with specific d UTP ase activity. d UTP ases have a central role in the regulation of d UTP intracellular levels and d TTP nucleotide metabolism. DR 2231 presents a conserved dimetal catalytic site, similar to all‐α dimeric d UTP ases, but contrary to these enzymes, it is unable to process d UDP . 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 d UMP or a d UTP 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. β‐ 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 d UDP β‐phosphate for hydrolysis explains DR 2231 preference towards d UTP . 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 .