8RPS

Crystal structure of human DNPH1 mutant- D80N bound to 5hmdUMP

Summary for 8RPS

• Entry DOI: 10.2210/pdb8rps/pdb
• Descriptor: 2'-deoxynucleoside 5'-phosphate N-hydrolase 1, 5-HYDROXYMETHYLURIDINE-2'-DEOXY-5'-MONOPHOSPHATE (3 entities in total)
• Functional Keywords: deoxyribonucleoside 5'-monophosphate n-glycosidase activity, hydrolase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 3
• Total formula weight: 49256.12

Authors

Devi, S.,da Silva, R.G. (deposition date: 2024-01-17, release date: 2024-11-20)

Primary citation

Carberry, A.E.,Devi, S.,Harrison, D.J.,da Silva, R.G.
Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: The Catalytic Roles of Tyr24 and Asp80.
Chembiochem, 25:e202400047-e202400047, 2024

PubMed Abstract: The human enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (HsDNPH1) catalyses the hydrolysis of 5-hydroxymethyl-2'-deoxyuridine 5'-phosphate to generate 5-hydroxymethyluracil and 2-deoxyribose-5-phosphate via a covalent 5-phospho-2-deoxyribosylated enzyme intermediate. HsDNPH1 is a promising target for inhibitor development towards anticancer drugs. Here, site-directed mutagenesis of conserved active-site residues, followed by HPLC analysis of the reaction and steady-state kinetics are employed to reveal the importance of each of these residues in catalysis, and the reaction pH-dependence is perturbed by each mutation. Solvent deuterium isotope effects indicate no rate-limiting proton transfers. Crystal structures of D80N-HsDNPH1 in unliganded and substrate-bound states, and of unliganded D80A- and Y24F-HsDNPH1 offer atomic level insights into substrate binding and catalysis. The results reveal a network of hydrogen bonds involving the substrate and the E104-Y24-D80 catalytic triad and are consistent with a proposed mechanism whereby D80 is important for substrate positioning, for helping modulate E104 nucleophilicity, and as the general acid in the first half-reaction. Y24 positions E104 for catalysis and prevents a catalytically disruptive close contact between E104 and D80.

PubMed: 38350003
DOI: 10.1002/cbic.202400047

Experimental method

X-RAY DIFFRACTION (1.75 Å)