PMC

The active site of λ phosphatase. Stereo view of the active site of λ-Pase (protomer C) from the crystal structure of Voegtli et al. () (PDB accession code 1G5B). The amino acid side chains coordinating the binuclear metal cluster and the sulfate ion are shown. The corresponding amino acids of CthPnkp are indicated in parentheses. The manganese ions are colored magenta. Water is colored red.

Mutational effects on 2′,3′ cyclic phosphodiesterase activity. Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 5 mM 2′,3′ cAMP, 1 U CIP and 0.5 µg CthPnkp as specified were incubated for 30 min at 45°C. (A) Effects of alanine mutations. (B) Effects of conservative substitutions. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown.

Phosphodiesterase activity of λ-Pase. (A) Reaction mixtures (25 µl) containing 0.2 mM MnCl₂, 10 mM DTT and either 100 mM Tris–HCl pH 8.0 and 10 mM p-nitrophenyl phosphate (filled square) or 100 mM Tris–HCl pH 7.0 and 10 mM bis-p-nitrophenyl phosphate (open circle) or p-nitrophenyl phenylphosphonate (filled circle), and λ-Pase as specified were incubated for 15 min at 37°C. (B) Reaction mixtures (25 µl) containing 100 mM Tris–acetate (filled circle) or Tris–HCl (open circle) at the pH specified, 0.5 mM MnCl₂, 10 mM DTT, 10 mM bis-p-nitrophenyl phosphate and 2 pmol λ-Pase were incubated for 10 min at 37°C. (C) Kinetics. Reaction mixtures (per 25 µl) containing 100 mM Tris–HCl (pH 7.0), 0.2 mM MnCl₂, 10 mM DTT, 10 mM bis-p-nitrophenyl phosphate and 2 pmol λ-Pase were incubated at 37°C. Two aliquots (25 µl) were withdrawn at the times specified, quenched immediately with EDTA and assayed for p-nitrophenol (filled circle) and inorganic phosphate (open circle).

Active sites of binuclear metallophosphodiesterases MJ0936 and Rv0805. Stereo views of the active site of Methanoccus jannaschii MJ0936 (top panel; PDB 1S3N) and Mycobacterium tuberculosis Rv0805 (bottom panel; PDB 2HY1) oriented similarly to the λ-Pase active site depicted in Figure 1. The amino acid side chains coordinating the binuclear metal cluster (and the phosphate ion in Rv0805) are shown. The metal ions are colored magenta. Water is colored red. Unlike CthPnkp and λ-Pase, the MJ0936 and Rv0805 phosphodiesterases have no arginine side chain in their active sites. Whereas the cyclic nucleotide phosphodiesterase Rv0805 has a phosphate-coordinating histidine corresponding to CthPnkp His264, the analogous residue is asparagine in MJ0936, which is unable to hydrolyze cyclic nucleotides. The water-mediated metal contact of Asp202 in λ-Pase is replaced by a direct interaction of the metal with a histidine in both MJ0936 and Rv0805.

Requirements for hydrolysis of p-nitrophenyl phenylphosphonate. (A) Reaction mixtures (25 µl) containing 50 mM Tris–acetate (filled circle) or Tris–HCl (open circle) at the pH specified, 0.5 mM MnCl₂, 10 mM p-nitrophenyl phenylphosphonate and 1 µg wild-type CthPnkp were incubated for 30 min at 45°C. (B) Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.0), 10 mM p-nitrophenyl phenylphosphonate, 1 µg of CthPnkp and either no divalent cation (lane –) or 0.5 mM MnCl₂, NiCl₂, CoCl₂, CdCl₂, CaCl₂, MgCl₂, ZnCl₂ or CuCl₂ were incubated for 30 min at 45°C. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown. (C) Reaction mixtures (350 µl) containing 50 mM Tris–HCl (pH 7.0), 10 mM p-nitrophenyl phenylphosphonate, 0.5 mM MnCl₂ and 7 µg CthPnkp were incubated at 45°C. Two aliquots (25 µl) were withdrawn at the times specified, quenched immediately with EDTA, and assayed for p-nitrophenol (filled circle) and inorganic phosphate (open circle). (D and E) Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.0), 0.5 mM MnCl₂, 10 mM p-nitrophenyl phenylphosphonate and 0.5 µg wild-type or mutant CthPnkp as specified were incubated for 30 min at 45°C. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown.

Substrate specificity of CthPnkp. Reaction mixtures (25 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 1.5 µg of CthPnkp and 10 mM substrate as specified were incubated for 30 min at 45°C. The reactions were quenched by adding 20 mM EDTA and then 0.9 ml of 1 M Na₂CO₃. Release of p-nitrophenol was determined by measuring A₄₁₀ and interpolating the value to a p-nitrophenol standard curve. The extents of formation of p-nitrophenol are plotted at left. The chemical structures of the substrates bis-p-nitrophenyl phosphate ①, p-nitrophenyl phenylphosphonate ②, dimethyl-p-nitrophenyl phosphate ③, thymidine 5′-monophosphate-p-nitrophenyl ester ④ and p-nitrophenyl phosphorylcholine ⑤ are shown at right.

Cyclic phosphodiesterase activity of λ-Pase. (A) Reaction mixtures (10 µl) containing 100 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM DTT, 10 mM substrate as specified and either 32 pmol λ-Pase or 1 U CIP (where indicated by +) were incubated for 30 min at 37°C. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown. (B) Reaction mixtures (per 10 µl) containing 100 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM DTT, 10 mM 2′,3′ cAMP and 64 pmol λ-Pase were incubated at 37°C. Aliquots (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. One micro liter of each sample was applied to a cellulose-F TLC plate. Markers 2′-AMP, 3′-AMP and 2′,3′ cAMP (5 nmol each) were spotted in lane M. The TLC plate was developed with buffer containing saturated ammonium sulfate/3 M sodium acetate/isopropanol (80/6/2). The nucleotides were visualized by photography under UV light.

Hydrolysis of cyclic phosphodiester substrates by CthPnkp. (A) Reaction mixtures (10 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM substrate as specified and either 4 µg wild-type CthPnkp, 1.4 µg CthPnkp-H189D, or calf intestine phosphatase (CIP; 1 U) where indicated by + were incubated for 30 min at 45°C. The reactions were quenched by adding EDTA (20 mM final concentration) and then 1 ml of malachite green reagent. Release of phosphate was determined by measuring A₆₂₀ and interpolating the value to a phosphate standard curve. Each datum in the bar graph is the average of two separate experiments. S.E. bars are shown. (B) Kinetics. Reaction mixtures (per 10 µl) containing 50 mM Tris–HCl (pH 7.5), 0.5 mM MnCl₂, 10 mM 2′,3′ cGMP and 1.4 µg of CthPnkp-H189D were incubated at 45°C. Samples (10 µl) were withdrawn at the times specified and quenched immediately with EDTA. Aliquots (1 µl) of each sample were applied to a cellulose-F TLC plate (EMD chemicals). Markers 2′,3′ cGMP and 3′GMP (5 nmol each) were spotted in lane M. The TLC plate was developed with buffer containing saturated ammonium sulfate/3 M sodium acetate/isopropanol (80/6/2). The nucleotides were visualized by photography under UV light.