Characterization of a family I inorganic pyrophosphatase from Legionella pneumophila Philadelphia 1

Abstract

Inorganic pyrophosphate (PP_i) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PP_i must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PP_i into two orthophosphates (P_i), preventing the toxic accumulation of the PP_i byproduct in cells and making P_i available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Å resolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) β-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg²⁺ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.

Keywords: Legionella pneumophila; Legionnaires' disease; SSGCID; Seattle Structural Genomics Center for Infectious Disease; inorganic pyrophosphatases; structural genomics.

Figure 1

Crystal structure of L. pneumophila PPase (LpPPase). (a) The homohexameric structure of LpPPase, which is a dimer of trimers, is shown. Individual monomers of the upper and lower trimer are colored in three different shades of green. All subunits are shown as ribbons. (b) One trimer is shown in surface representation, while the other is shown as ribbons. Individual subunits colored as in (a) demonstrating symmetry. (c) Representative monomer of LpPPase annotated with secondary-structure elements including β-sheets (β), α-helices (α) and 3₁₀-helices (η). (d) Secondary-structure elements involved in the quaternary-structure interfaces for each monomer are colored red and labeled, with the trimer–trimer interface boxed.

Figure 2

Sequence and structural alignments of LpPPase. (a) Primary-sequence alignment of L. pneumophila PPase (LpPPase; PDB entry 6n1c) with PPases from A. baumannii (PDB entry 6k21), E. coli (PDB entry 1obw), O. antarctica (PDB entry 3i4q) and P. aeruginosa (PDB entry 4xel). Secondary-structure elements of LpPPase are shown: β-sheets (β), α-helices (α), 3₁₀-helices (η), β-turns (TT) and α-turns (TTT). Identical residues are shown in white on a black background, while conserved residues are shown in bold and related residues are boxed. Pink diamonds indicate catalytically significant residues and blue diamonds indicate residues that bind the substrate. (b) LpPPase (green) aligned with EcPPase from E. coli (gray; PDB entry 1obw). Labeled residues shown as sticks are important for catalysis (pink) or for substrate binding (blue), corresponding to the diamonds in (a). (c) LpPPase (green) aligned with YPPase from S. cerevisiae (purple; PDB entry 1e6a), a eukaryotic family I PPase.

Figure 3

SEC analysis of LpPPase. (a) Two concentrations of LpPPase were investigated using size-exclusion chromatography (SEC), with elution volumes (V _e) indicated by the dotted gray lines. LpPPase had a V _e of 77.03 ml at 0.5 mg ml⁻¹ (solid green line) and 72.25 ml at 7.0 mg ml⁻¹ (dashed green line) from a HiLoad 16/60 Superdex 200 pg SEC column. (b) The native molecular weights (M _r) at each concentration (green circles) were estimated using a calibration curve (black circles). (c) The table shows the partition coefficients (K _av), individual elution volumes (V _e) and the known or calculated molecular weights (M _r) of each protein. The calculated M _r of LpPPase indicates likely trimeric (0.5 mg ml⁻¹) and hexameric (7.0 mg ml⁻¹) forms. The predicted monomeric weight of LpPPase is given in the last row for reference.

Figure 4

LpPPase enzymatic activity. (a) LpPPase enzymatic activity on p-nitrophenyl phosphate (pNPP) using Mg²⁺ as a metal cofactor. The curve was fitted with the Michaelis–Menten equation, which gave a K _m of 69.7 ± 18.8 mM and a V _max of 2.36 ± 0.27 nmol ml⁻¹ min⁻¹. (b) LpPPase activity was investigated with four alternative metal cofactors, Mn²⁺, Zn²⁺, Co²⁺ and Ca²⁺, using the pNPP concentration where V _max was previously reached. Inhibition by F⁻ in the presence of Mg²⁺ was also tested using sodium fluoride as a source of fluoride ions. The reaction velocities shown in (b) were normalized to V _max in (a), which was measured with Mg²⁺ as the metal cofactor. Error bars show standard deviations. Reactions were run in duplicate. n.d., not detected.