Journal Article

Structural dynamics of human deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase)

Dobrovolska, Olena, Sarre, Ravdna, Lundström, Patrik, Turcu, Diana, Agback, Tatiana, Halskau, Øyvind, Isaksson, Johan

2024

Overview

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2’-deoxyuridine 5’-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2’-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

• Publisher: Nature Publishing Group UK,Nature Portfolio
• Subject: 631/45/173 / 631/535/878/1263 / Allosteric Regulation / Biochemistry and Molecular Biology / Biokemi och molekylärbiologi / Human dUTPase / Humanities and Social Sciences / Humans / Kinetics / Molecular Dynamics Simulation / Multidimensional NMR / multidisciplinary / Protein Binding / Protein Conformation / Protein dynamics / Pyrophosphatases - chemistry / Pyrophosphatases - metabolism / Science / Science (multidisciplinary) / Substrate Specificity