Direct contacts between conserved motifs of different subunits provide major contribution to active site organization in human and mycobacterial dUTPases

Abstract 

dUTP pyrophosphatases (dUTPases) are essential for genome integrity. Recent results allowed characterization of the role of conserved residues. Here we analyzed the Asp/Asn mutation within conserved Motif I of human and mycobacterial dUTPases, wherein the Asp residue was previously implicated in Mg²⁺-coordination. Our results on transient/steady-state kinetics, ligand binding and a 1.80Å resolution structure of the mutant mycobacterial enzyme, in comparison with wild type and C-terminally truncated structures, argue that this residue has a major role in providing intra- and intersubunit contacts, but is not essential for Mg²⁺ accommodation. We conclude that in addition to the role of conserved motifs in substrate accommodation, direct subunit interaction between protein atoms of active site residues from different conserved motifs are crucial for enzyme function.

Abbreviations: M. tuberculosis, Mycobacterium tuberculosis, dUTP, 2′-deoxyuridine triphosphate, dUTPase, dUTP pyrophosphatase, dUPNPP, α,β-imido-dUTP, hDUT, human dUTPase, mtDUT, Mycobacterium tuberculosis dUTPase, hDUT^D49N,F158W, Asp49Asn/Phe158Trp double mutant construct of human dUTPase, mtDUT^D28N, mtDUT^T138STOP, and mtDUT^D28N,H145W, Asp28Asn, Thr138STOP and Asp28Asn/His145Trp mutant constructs of Mycobacterium tuberculosis dUTPase

Keywords: dUTPase, Mycobacterium tuberculosis, DNA repair, Nucleotide hydrolysis, Aspartate, Asparagine, Oligomer