Hexamer structure of Pseudomonas aeruginosa deoxynucleotide triphosphate triphosphohydrolase and its involvement in catalysis and DNA binding

TL;DR

This paper studies the structure and function of a bacterial enzyme that helps regulate DNA building blocks and may interact with DNA during replication.

Contribution

The study reveals the hexameric structure of paDT and identifies its active site and DNA-binding region for the first time.

Findings

paDT forms a hexamer with a conserved metal-binding motif in the interdomain dent.

paDT binds single-stranded DNA via a positively charged patch in the intersubunit cleft.

The interdomain dent is proposed to function as the active site for dNTP hydrolysis.

Abstract

Genetic mutations often have lethal consequences for cells, occurring more frequently when the pool of deoxynucleotide triphosphates (dNTPs) is imbalanced. To keep the dNTP pool balanced, intracellular dNTP levels are tightly regulated through dNTP biosynthesis and degradation pathways. dNTP triphosphohydrolase contributes to dNTP homeostasis and the fidelity of DNA replication by hydrolyzing dNTP into 2'- deoxyribonucleoside and inorganic triphosphate. In this study, we conducted structural analysis to provide insight into the dNTP hydrolysis mechanism used by the dNTP triphosphohydrolase of the opportunistic pathogen Pseudomonas aeruginosa (paDT). paDT consists of three domains and assembles into a hexamer as a characteristic quaternary structure of phylogenetic clade A of dNTP triphosphohydrolases. Each subunit of the paDT hexamer accommodates a metal ion in an interdomain dent using a conserved motif composed of histidine and aspartate residues. This observation and our comparative analyses suggest that the interdomain dent functions as the active site of paDT. Interestingly, paDT showed ssDNA-binding ability, presumably via a positively charged patch located in the intersubunit cleft, allowing us to propose that paDT recognizes ssDNA as an allosteric regulator.