Metal ion coordination in the E. coli Nudix hydrolase dihydroneopterin triphosphate pyrophosphatase: New clues into catalytic mechanism.

Shannon E Hill,Stephen Quirk,Elaine Nguyen,Dana M Freeman,Chiamaka U Ukachukwu,Raquel L Lieberman,Titus J Boggon

doi:10.1371/journal.pone.0180241

Shannon E Hill, Stephen Quirk + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0180241

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Abstract

Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.

Highlights

Enzymes involved in folate biosynthesis in bacteria are targets of antimicrobial agents that have been used in the clinic since the 1930s [1, 2]
We sought to crystallize DHNTPase in the presence of dihydroneopterin as a first step toward inhibitor design
Optimized crystals grown in ~35% PEG 8000, 0.05 mM ammonium sulfate were soaked in a range of dihydroneopterin concentrations and either 10 mM MgSO4, NiCl2, or 5 mM CoCl2

Summary

Introduction

Enzymes involved in folate biosynthesis in bacteria are targets of antimicrobial agents that have been used in the clinic since the 1930s [1, 2]. The metallated structures reveal a tetranuclear metal binding site coordinated by enzyme residues within and outside of the Nudix signature motif, water molecules, and a sulfate anion used during crystallization.

Results

Conclusion