Abstract
Human NUDT16 is a member of the NUDIX hydrolase superfamily. After having been initially described as an mRNA decapping enzyme, recent studies conferred it a role as an “housecleaning” enzyme specialized in the removal of hazardous (deoxy)inosine diphosphate from the nucleotide pool. Here we present the crystal structure of human NUDT16 both in its apo-form and in complex with its product inosine monophosphate (IMP). NUDT16 appears as a dimer whose formation generates a positively charged trench to accommodate substrate-binding. Complementation of the structural data with detailed enzymatic and biophysical studies revealed the determinants of substrate recognition and particularly the importance of the substituents in position 2 and 6 on the purine ring. The affinity for the IMP product, harboring a carbonyl in position 6 on the base, compared to purine monophosphates lacking a H-bond acceptor in this position, implies a catalytic cycle whose rate is primarily regulated by the product-release step. Finally, we have also characterized a phenomenon of inhibition by the product of the reaction, IMP, which might exclude non-deleterious nucleotides from NUDT16-mediated hydrolysis regardless of their cellular concentration. Taken together, this study details structural and regulatory mechanisms explaining how substrates are selected for hydrolysis by human NUDT16.
Highlights
Multiple non-canonical nucleotides appear as substrates and products in nucleotide metabolism, while multiple other damaged forms can be present as a consequence of various types of cellular stress
Upon inspection of the interactions between symmetry-related molecules, it is clear that NUDT16 exists as a dimer (Fig 1A and 1B), which is common for NUDIX family members
This observation and the analysis of potential protein-protein interfaces as determined by the PISA server confirm that NUDT16 is present as a dimer in solution [22]
Summary
Multiple non-canonical nucleotides appear as substrates and products in nucleotide metabolism, while multiple other damaged forms can be present as a consequence of various types of cellular stress. The appearance of elevated concentrations of any of these nucleotides may alter normal cellular function through competition with canonical nucleotides for binding to PLOS ONE | DOI:10.1371/journal.pone.0131507. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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