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

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

Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution

Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), which is a molecule secreted by Streptomyces scabies, the plant pathogen responsible for potato scab. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis in vitro, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but is readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expand our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

DHNTPase metal cluster.

<p>(a) Left: Final 2Fo-Fc electron density map (blue) for Co-bound DHNTPase contoured at 1 sigma superimposed with Fo-Fc map (green) contoured at 3 σ (generated by using only the final polypeptide chain) and an anomalous Fourier map (yellow) contoured at 5 σ. Right: Fo-Fc and anomalous maps only. (b) Superposition of Ni-bound DHNTPase (magenta) and Co-bound DHNTPase (cyan) zoomed into the metal binding site. Dashed lines are selected interactions ≤2.5 Å from protein, sulfate, and/or metal. (c) Chemdraw representation of all ~2.5 Å interactions observed crystallographically in the Co²⁺-bound structure. Numbered metals are referenced in text. W = water. (d) Superposition of DHNTPase solved previously with PPi (orange stick) and Na⁺ (purple ball) from PDB code 2O5W chain A (pale yellow cartoon) and Co-bound DHNTPase (cyan) zoomed into metal centers. (e) Left: Superposition of Ap4A (light green) and Co-bound DHNTPase (cyan) zoomed into metal centers. Both structures contain four metals and a bound sulfate or phosphate. Middle: Comparison of metal binding region of RppH (orange) and Co-bound DHNTPase. RppH was crystallized with three metals overlapping with M1-3 in our structure; the DHNTPase sulfate overlays well with the pyrophosphate leaving group of the cocrystallized substrate analog in RppH. Right: Superposition of all three structures highlighting key coordinating residues to metal and substrate. All three structures have a modeled solvent molecule in line with a putative catalytic base. A unique feature of DHNTPase in this region is involvement of Lys7 at the N-terminus.</p

Thermodynamic parameters for metals binding to DHNTPase.

<p>Thermodynamic parameters for metals binding to DHNTPase.</p

Thermodynamic parameters for SO₄^2- binding to DHNTPase.

<p>Thermodynamic parameters for SO₄^2- binding to DHNTPase.</p

Biochemical characterization of metal binding to DHNTPase.

<p>(a) Enzyme assays with varying metals (left) and dose dependence with increasing concentrations of Mg²⁺. (b) Typical ITC binding analysis between DHNTPase and Co²⁺ (left) and Ni²⁺ (right). (c) Fraction unfolded DHNTPase as a function of temperature. Thermal CD scans were performed as described in Methods. Open triangles, Wild-type apo DHNTPase; Closed circles, DHNTPase/SO₄^2-/Ni²⁺; Open circles, DHNTPase/SO₄^2-/Co²⁺; Closed triangles, DHNTPase/SO₄^2-. (d) Typical ITC binding analysis between apo DHNTPase and SO₄^2-. Thermodynamic parameters for ITC experiments in (b) and (d) are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180241#pone.0180241.t004" target="_blank">Table 4</a>.</p

Calculated T_m values for DHNTPase and DHNTPase-metal complexes from CD melt.

<p>The value shown is the mean of three replicate experiments. The number in parentheses is the standard deviation.</p

Substrate docking model.

<p>(a) Binding pocket for dihydroneopterin moiety with key predicted polar interactions presented in dashed lines and hydrophobic residues presented as sticks. Metals M1-4 are presented as balls but were not present during docking calculation. (b) Chemdraw representation of predicted interaction between triphosphate moiety with key stabilizing interactions with four metals and protein residues. Waters were excluded from docking calculation. Crystallographic water predicted to be activated for catalysis (green) is 2.6 Å from predicted scissile P-O bond in substrate. W = water.</p

Overall structure and sequence comparison among DHNTPase structures and other Nudix hydrolases.

<p>(a) Superposition of apo DHNTPase (green), Ni-bound (magenta), and Co-bound (blue) structures from this study. Sulfates are presented as sticks, and balls represent metals. (b) Superposition of apo DHNTPase with two distinct monomers from PDB code 201C (yellow, beige). (c) Superposition of Co-bound structure (blue) with close structural homologs 1KTG (light green) and 4S2Y (orange). Sulfate from Co-bound structure and phosphate from 1KTG are presented as sticks, and balls represent metals. (d) Multiple sequence alignment of DHNTPase with representative Nudix hydrolases RppH, Ap4A, and MutT. Similar residues are boxed and red font. Identical residues are white with red background. Secondary structure of DHNTPase presented on top of the sequence. β-strands: arrows, helices: coils, T: turns.</p