8OS9

Structure of Homo sapiens 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1)

Summary for 8OS9

• Entry DOI: 10.2210/pdb8os9/pdb
• Descriptor: 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (2 entities in total)
• Functional Keywords: deoxyribonucleoside 5'-monophosphate n-glycosidase activity, hydrolase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 4
• Total formula weight: 64776.88

Authors

Devi, S.,da Silva, R.G. (deposition date: 2023-04-18, release date: 2023-08-30, Last modification date: 2023-09-13)

Primary citation

Devi, S.,Carberry, A.E.,Zickuhr, G.M.,Dickson, A.L.,Harrison, D.J.,da Silva, R.G.
Human 2'-Deoxynucleoside 5'-Phosphate N -Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.
Biochemistry, 62:2658-2668, 2023

PubMed Abstract: The enzyme 2'-deoxynucleoside 5'-phosphate -hydrolase 1 (DNPH1) catalyzes the -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 /, two groups with an average p of 6.4 must be deprotonated, while two groups with an average p 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 /. Solvent deuterium isotope effects on / and were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

PubMed: 37582341
DOI: 10.1021/acs.biochem.3c00369

Experimental method

X-RAY DIFFRACTION (1.7 Å)