Archaeal Inorganic Pyrophosphatase Displays Robust Activity under High-Salt Conditions and in Organic Solvents

Abstract

Soluble inorganic pyrophosphatases (PPAs) that hydrolyze inorganic pyrophosphate (PPi) to orthophosphate (Pi) are commonly used to accelerate and detect biosynthetic reactions that generate PPi as a by-product. Current PPAs are inactivated by high salt concentrations and organic solvents, which limits the extent of their use. Here we report a class A type PPA of the haloarchaeon Haloferax volcanii (HvPPA) that is thermostable and displays robust PPi-hydrolyzing activity under conditions of 25% (vol/vol) organic solvent and salt concentrations from 25 mM to 3 M. HvPPA was purified to homogeneity as a homohexamer by a rapid two-step method and was found to display non-Michaelis-Menten kinetics with a Vmax of 465 U · mg(-1) for PPi hydrolysis (optimal at 42°C and pH 8.5) and Hill coefficients that indicated cooperative binding to PPi and Mg(2+). Similarly to other class A type PPAs, HvPPA was inhibited by sodium fluoride; however, hierarchical clustering and three-dimensional (3D) homology modeling revealed HvPPA to be distinct in structure from characterized PPAs. In particular, HvPPA was highly negative in surface charge, which explained its extreme resistance to organic solvents. To demonstrate that HvPPA could drive thermodynamically unfavorable reactions to completion under conditions of reduced water activity, a novel coupled assay was developed; HvPPA hydrolyzed the PPi by-product generated in 2 M NaCl by UbaA (a "salt-loving" noncanonical E1 enzyme that adenylates ubiquitin-like proteins in the presence of ATP). Overall, we demonstrate HvPPA to be useful for hydrolyzing PPi under conditions of reduced water activity that are a hurdle to current PPA-based technologies.

FIG 1

Evolutionary relationships of archaeal inorganic pyrophosphatases of the IPR008162 family. A phylogenetic tree of amino acid sequences was used to represent the evolutionary relationships of archaeal PPAs. Archaeal PPAs biochemically characterized are highlighted (●), including Thermoplasma acidophilum TaPPA (Ta0399) (4, 35, 36), Pyrococcus horikoshii PhPPA (PH1907) (5, 37–39), Sulfolobus sp. StPPA (STK_05240) and SaPPA (Saci_0955) (40–44), Methanobacterium thermoautotrophicum MtPPA (MTH_263) (45), Thermococcus thioreducens TtPPA (H0USY5_9EURY) (46), and H. volcanii HvPPA (HVO_0729) (this study). The optimal tree with the sum of branch lengths of 15.65877199 is represented. The tree is drawn to scale, with branch lengths in the same units as those for the evolutionary distances used to infer the phylogenetic tree. See Materials and Methods for details.

FIG 2

Structural comparison of class A type inorganic pyrophosphatases. (A) Multiple-amino-acid-sequence alignment of PPAs. Highlighted are identical (black), functionally similar (gray), Cys-X₆₃-Cys motif (blue), and conserved active-site (red) residues. Predicted α-helix and β-sheet structures and Asp residues (*) coordinating Mg²⁺/Mn²⁺ are indicated above the alignment. Abbreviations are as in Fig. 1. (B) 3D structural comparison of PPAs. A 3D structural model of HvPPA (blue ribbon) compared to the X-ray crystal structures of PhPPA (PDB 1UDE) (tan ribbon), SaPPA (PDB 1QEZ), TtPPA (PDB 3R5U), and Saccharomyces cerevisiae ScPPA (PDB 1E9G) is shown. Mn²⁺ ions (purple ball), phosphate ions (orange and red stick), and water (red ball) ligands are overlaid onto the 3D model. C-terminal (Ct) and N-terminal (Nt) residues are indicated. HvC24 and HvC85 are cysteine residues conserved in all haloarchaeal PPAs. Conserved active-site residues analogous to ScPPA include HvK31 (ScK56), HvE33 (ScE58), HvR45 (ScR78), HvY57 (ScY93), HvD67 (ScD115), HvD69 (ScD117), HvD72 (ScD120), HvD99 (ScD147), HvD104 (ScD152), HvK106 (ScK154), HvY141 (ScY192), and HvK142 (ScK193). HvD67, HvD72, and HvD104 are predicted to coordinate the Mg²⁺ and Mn²⁺ ions. the HvPPA 3D structure was modeled by Phyre2 intensive mode at a confidence of >90% accuracy for 175 out of 177 residues (99%). (C) Comparison of electrostatic potentials of PPAs. Electrostatic potential is represented by coulombic surface coloring with the unit of the potential colored in the range of values −10 (red), 0 (white), and 10 (blue) kcal/mol · e using Chimera v 1.7.

FIG 3

Class A type inorganic pyrophosphatase purified from Haloferax volcanii by tandem affinity and size exclusion chromatography. (A) HvPPA fractions analyzed by SDS-PAGE. Lanes 1 and 2, H. volcanii H26 (lane 1) and H26-pJAM2920 expressing His₆-HvPPA (lane 2) applied at an OD₆₀₀ of 0.065 cell per lane. Lanes 3 and 4, Ni²⁺-Sepharose (lane 3) and Superdex 200 GL10/300 (lane 4) chromatography fractions of HvPPA applied at 1 μg protein per lane. Protein was separated by reducing 10% SDS-PAGE and analyzed by Coomassie blue R-250 staining (upper panel) and anti-His₆ immunoblotting (lower panel). (B) HvPPA analyzed by Superdex 200 30/100 GL size exclusion chromatography. Column fractions are represented by a semilog plot of molecular mass versus K_av, with molecular mass standards (●) and HvPPA hexamer (□) (134 kDa) and trimer (○) (64 kDa) indicated.

FIG 4

Effect of pH, temperature, salt, and divalent cations on Haloferax volcanii inorganic pyrophosphatase (HvPPA) activity. (A to C) HvPPA was equilibrated for 10 min at the pH, temperature, and NaCl concentrations indicated prior to addition of PP_i (0.25 mM). MgCl₂ (2.5 mM) was included in the activity assay mixtures. For pH optimum assays, reactions were in 3 M NaCl with 20 mM buffer (sodium acetate [pH 4 to 5], morpholineethanesulfonic acid [MES] [pH 6 to 6.5], Tris-Cl [pH 7 to 9], and N-cyclohexyl-3-aminopropanesulfonic acid [CAPS] [pH 10]). For temperature optimum assays, reactions were in 20 mM Tris-Cl (pH 8) buffer containing 3 M NaCl. For salt optimum assays, HvPPA was diluted into 20 mM Tris-Cl (pH 8) buffer with NaCl concentrations as indicated. (D) To test the influence of cations, HvPPA was dialyzed sequentially against 500 ml of buffer (20 mM Tris-Cl [pH 8], 2 M NaCl, and 1 μM EDTA) (4 h at 4°C) and the same buffer with EDTA omitted (4 h at 4°C). Reaction mixtures for panel D contained HvPPA (0.93 μg), 20 mM Tris-Cl (pH 8), 2 M NaCl, 0.25 mM PP_i, and divalent metal at the concentration indicated. Metals used were CaCl₂ · 2H₂O, ZnCl₂, CoCl₂ · 6H₂O, MnCl₂ · 4H₂O, NiCl₂ · 6H₂O, and MgCl₂ · 6H₂O. (E) Non-EDTA-treated HvPPA was assayed in 20 mM Tris-Cl (pH 8), 2 M NaCl, 0.25 mM PP_i, and MgCl₂ at the concentrations indicated. Reactions were monitored for 10 min at room temperature unless otherwise indicated. One hundred percent activity is relative to highest reported within each panel.

FIG 5

Sodium fluoride-based inhibition of Haloferax volcanii inorganic pyrophosphatase (HvPPA). (A) HvPPA (1.3 μg) was assayed in a 1-ml reaction volume containing 1 mM PP_i, 10 mM MgCl₂, 3 M NaCl, and 20 mM Tris-Cl buffer (pH 7.5) supplemented with NaF as indicated. Reactions were monitored for 15 min at room temperature. (B) Amino acid residues of HvPPA predicted to interact with F⁻-, PP_i-, H₂O-, and Mg²⁺-bound molecules as determined by modeling compared to the X-ray crystal structure of E. coli PPA (PDB 2AUU).

FIG 6

Effect of salt, solvent, and temperature on Haloferax volcanii inorganic pyrophosphatase (HvPPA). (A) HvPPA thermostability. HvPPA was incubated at 42°C and 65°C as indicated with enzyme at 0.093 mg per ml of buffer (20 mM Tris-Cl [pH 8], 2.5 mM MgCl₂, and 3 M NaCl). HvPPA was diluted to 0.28 μg per 100 μl buffer (with MgCl₂ increased to 10 mM) and assayed by addition of 1 mM PP_i substrate (10 min, room temperature). Activity is relative to that of samples incubated on ice. (B) HvPPA stability in 50% (vol/vol) solvent. HvPPA was incubated for 2 h (on ice) at 0.47 mg per ml buffer (20 mM Tris-Cl [pH 8] and 2 M NaCl) supplemented with solvent as indicated. HvPPA was diluted to 2.4 μg per 100 μl buffer (20 mM Tris-Cl [pH 8], 2.5 mM MgCl₂, and 2 M NaCl) and assayed by addition of 50 μM PP_i substrate (10 min, room temperature). Activity is relative to that of samples incubated with no solvent. (C) HvPPA activity in 25% (vol/vol) solvent. Reaction mixtures were 500 μl with 2.8 μg HvPPA, 1.5 M NaCl, 1.5 mM PP_i, and 10 mM MgCl₂. Reaction was for 10 min at room temperature. (D) HvPPA stability in salt. HvPPA was incubated for 2 h (on ice) at 0.3 mg per ml of buffer (20 mM Tris-Cl, pH 8) supplemented with NaCl at the concentrations indicated. HvPPA was diluted to 0.87 μg per 100 μl reaction buffer as for panel B. Activity is relative to that of samples incubated in buffer supplemented with 3 M NaCl.

FIG 7

Haloferax volcanii inorganic pyrophosphatase (HvPPA) coupled adenylation assay at high temperature and reduced water activity. (A) Schematic of the coupled assay. Adenylation of the ubiquitin-like SAMP by the E1-like enzyme UbaA was monitored by HvPPA-mediated hydrolysis of the PP_i (P₂O₇⁴⁻) by-product to 2P_i (2 mol HPO₄²⁻) at 42°C. (B) Generation of P_i correlated with the addition of ATP, UbaA, HvPPA, and SAMP1 to the assay buffer. ΔGG, C-terminal diglycine residue deletion.