Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : mechanism of 2'-deoxyuridine 5'-monophosphate hydrolysis
Abstract
The enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2′-deoxyuridine 5′-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2′-deoxynucleoside 5′-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2′-deoxyuridine 5′-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography–mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid–base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.
Citation
Devi , S , Carberry , A E , Zickuhr , G M , Dickson , A , Harrison , D J & da Silva , R G 2023 , ' Human 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 : mechanism of 2'-deoxyuridine 5'-monophosphate hydrolysis ' , Biochemistry , vol. 62 , no. 17 , pp. 2658-2668 . https://doi.org/10.1021/acs.biochem.3c00369
Publication
Biochemistry
Status
Peer reviewed
ISSN
0006-2960Type
Journal article
Description
Funding: Industrial Biotechnology Innovation Centre - 2021-01-01.Collections
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