Family II pyrophosphatases from photosynthetic bacteria can hydrolyze free pyrophosphate

Figures & data

Table 1 Specific activity of family I (Rsp. rubrum) and family II (Rba. sphaeroides, Rba. capsulatus, Rdv. sulfidophilum) PPases, with and without divalent cation added at the hydrolysis reaction medium: 50 mM Tris–HCl (pH 8.6), 2 mM Na4PPi, and 3 mM of divalent cation

Figure 1 EDTA effect on the hydrolysis of pyrophosphatase.

Notes: Reaction conditions were 50 mM Tris–HCl (pH 8.6) and EDTA at the concentrations indicated on the abscissa. Different complexes were used as substrates: Mg–PPi (3 mM MgCl₂ and 2 mM Na₄PPi) in Rsp. rubrum (•) family I sPPase and PPi 2 mM without divalent cation added in Rba. capsulatus (◼), Rba. sphaeroides (⧫), and Rdv. sulfidophilum (▴) family II sPPases. For family I pyrophosphatase, 100% is equivalent to 127 nmol Pi·min⁻¹ (mg of protein)⁻¹, and for family II pyrophosphatases, 100% is equivalent to 1,240 nmol Pi·min⁻¹ (mg of protein)⁻¹.
Abbreviations: EDTA, ethylenediaminetetraacetic acid; Rsp. rubrum, Rhodospirillum rubrum ;Rba. capsulatus, Rhodobacter capsulatus ;Rba. sphaeroides, Rhodobacter sphaeroides ;Rdv. sulfidophilum, Rhodovulum sulfidophilum.

Figure 2 EDTA effect on the Rba. sphaeroides recombinant sPPiase activity.

Notes: Black bars represent reactions with 3 mM MgCl₂ added, and white bars represent reactions in which no divalent cation was added. -EDTA represents hydrolysis reaction without EDTA. +EDTA represents hydrolysis reaction with 100 mM EDTA-preincubated enzyme for 15 min at room temperature. Dialyzed represents EDTA-preincubated enzyme dialyzed against 20 mM Tris–HCl, pH 8.6, and 0.1 mM CoCl₂. Mg²⁺ incubated represents dialyzed enzyme, incubated with 7 mM MgCl₂ for 7 days at 8°C. Reaction conditions were 50 mM Tris–HCl (pH 8.6), 2 mM Na4PPi, and EDTA. Reaction incubation temperature was 37°C.
Abbreviations: EDTA, ethylenediaminetetraacetic acid; Rba. sphaeroides, Rhodobacter sphaeroides.

Figure 3 Silver-stained native PAGE of the recombinant Rba. sphaeroides sPPase preincubated with different concentrations of EDTA.

Abbreviations: PAGE, polyacrylamide gel electrophoresis; Rba. sphaeroides, Rhodobacter sphaeroides; EDTA, ethylenediaminetetraacetic acid.