A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity

Abstract

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.

FIGURE 1.

Active sites of binuclear metallophosphodiesterases Rv0805 and YfcE. The top and middle panels show stereo views of the active sites of M. tuberculosis Rv0805 (top panel; Protein Data Bank code 2HY1) and E. coli YfcE (middle panel; Protein Data Bank code 1SU1, protomer D). The amino acid side chains coordinating the binuclear metal cluster and either the phosphate ion in Rv0805 or sulfate ion in YfcE are shown. The metal ions are colored magenta. Water is colored red. The phosphate-binding histidine in Rv0805 (His⁹⁸) is replaced by a cysteine in YfcE (Cys⁷⁴). The bottom panel shows models of two potential orientations of 2′,3′-cGMP in the active site of Rv0805. The 2′,3′-cGMP molecule was imported from Protein Data Bank code 1GSP. The cyclic phosphate was superimposed on the phosphate anion in the Rv0805 structure. When the ribose O2′ is apical to the metal-bridged water nucleophile (left), the reaction yields a 3′-PO₄ nucleotide product. When the ribose O3′ atom is apical to the water nucleophile (right), the product is a 2′-PO₄ nucleotide. The His⁹⁸ side chain is poised to donate a hydrogen bond to the leaving ribose oxygen atom in the modeled 2′,3′-cGMP substrate in either orientation.

FIGURE 2.

Purification of Rv0805 and YfcE. Aliquots (5 μg) of the nickelagarose preparations of Rv0805 and YfcE containing the indicated amino acids at positions 98 and 74, respectively, were analyzed by SDS-PAGE. The Coomassie Blue-stained gel is shown. The mobility and sizes (kDa) of marker polypeptides are indicated on the left.

FIGURE 3.

Phosphoesterase activities of recombinant Rv0805 proteins. A, hydrolysis of bis-p-nitrophenyl phosphate. Reaction mixtures (25 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm bis-p-nitrophenyl phosphate, and either wild type Rv0805, H98N, or H98A as specified (0.016, 0.031, 0.063, 0.125, or 0.25 μm enzyme) were incubated for 10 min at 37 °C. B, hydrolysis of p-nitrophenyl phosphate. Reaction mixtures (25 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm p-nitrophenyl phosphate, and either wild type Rv0805, H98N, or H98A as specified (1, 2, 4, 8, or 16 μm enzyme) were incubated for 10 min at 37 °C. C, hydrolysis of 2′,3′-cAMP. Reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm 2′,3′-cAMP and either wild type Rv0805, H98N, or H98A as specified (0.16, 0.31, 0.62, 1.25, or 2.5 μm enzyme) were incubated for 10 min at 37 °C. D, reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm of either 2′,3′-cAMP or 3′,5′-cAMP, 1 unit CIP, and wild type Rv0805 as specified (0.16, 0.31, 0.62, 1.25, or 2.5 μm enzyme for reactions containing 2′,3′-cAMP; 5, 10, or 20 μm enzyme for reactions containing 3′,5′-cAMP) were incubated for 10 min at 37 °C. The extents (nmol) of formation of p-nitrophenol (A and B) or inorganic phosphate (C and D) are plotted as a function of input enzyme (pmol). Each datum is the average of two separate experiments; mean absolute error bars are included for all points but may not be visible where the error is small.

FIGURE 4.

Hydrolysis of cyclic phosphodiester substrates by Rv0805. A, reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.0), 0.5 mm MnCl₂, 10 mm cNMP as specified, and Rv0805 (6.25 pmol; 0.625 μm) or CIP (1 unit) where indicated by + were incubated for 15 min at 37 °C. Each datum for P_i formation is the average of two separate experiments; mean absolute error bars are shown. B, product analysis. Reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm 2′,3′-cAMP, and wild type Rv0805 as specified (corresponding to 0, 0.62, 1.25, 2.5, 5.0, or 10 μm enzyme) were incubated for 5 min at 37 °C. The reactions were quenched with EDTA. Aliquots (1 μl) of each sample were applied to a cellulose-F TLC plate (EMD chemicals). Ascending TLC was performed with buffer containing saturated ammonium sulfate, 3 m sodium acetate, isopropanol (80/6/2). The nucleotides were visualized by photography under UV light. The positions of the 2′,3′-cAMP, 3′-AMP and 2′-AMP standards are indicated on the right.

FIGURE 5.

Phosphoesterase activities of recombinant YfcE proteins. A, hydrolysis of bis-p-nitrophenyl phosphate. Reaction mixtures (25 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm bis-p-nitrophenyl phosphate, and either wild type YfcE, C74H, C74N, or C74A as specified (0.016, 0.031, 0.062, 0.125, or 0.25 μm enzyme) were incubated for 10 min at 37 °C. B, hydrolysis of p-nitrophenyl phosphate. Reaction mixtures (25 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm p-nitrophenyl phosphate, and either wild type YfcE, C74H, C74N, or C74A as specified (0.25, 0.5, 1, 2, or 4 μm enzyme) were incubated for 10 min at 37 °C. C, hydrolysis of 2′,3′-cAMP. Reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm 2′,3′-cAMP, and either wild type, C74H, C74N, or C74A as specified (0.625, 1.25, 2.5, 5, or 10 μm enzyme) were incubated for 15 min at 37 °C. The extents (nmol) of formation of p-nitrophenol (A and B) or inorganic phosphate (C) are plotted as a function of input enzyme (pmol). Each datum is the average of two separate experiments; mean absolute error bars are included for all points but may not be visible where the error is small.

FIGURE 6.

Hydrolysis of cyclic phosphodiester substrates by YfcE-C74H. A, reaction mixtures (10 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm substrate as specified, and YfcE-C74H (25 pmol; 2.5 μm enzyme) or CIP (1 unit) where indicated by + were incubated for 15 min at 37 °C. Each datum for P_i formation is the average of two separate experiments; mean absolute error bars are included for all data but may not be visible where the error is small. B, product analysis. A reaction mixture (90 μl) containing 50 mm Tris-HCl (pH 8.5), 0.5 mm MnCl₂, 10 mm 2′,3′-cAMP, and 45 μg of YfcE-C74H (13.5 μm enzyme) were incubated at 37 °C. Aliquots (10 μl) were withdrawn at the times specified and quenched immediately with EDTA. 1 μl 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. TLC was performed as described in Fig. 4. The nucleotides were visualized by photography under UV light.