Bacillus cereus phosphopentomutase is an alkaline phosphatase family member that exhibits an altered entry point into the catalytic cycle

Abstract

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn(2+)-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[(18)O(3)]phosphate and [U-(13)C(5)]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

FIGURE 1.

Generalized catalytic cycle for alkaline phosphatase superfamily proteins. For all previously characterized alkaline phosphatase family members, the catalytic cycle begins with a dephosphorylated (or desulforylated) enzyme, E-OH (state 1). Donor substrate (R_D-O-X, where X represents PO₃H⁻, SO₃H⁻) binds and is attacked by the catalytic nucleophile (states 2 and 3). The dephosphorylated donor substrate (R_D-OH) is released, leaving a covalently modified enzyme intermediate (E-O-X) (state 4). An acceptor substrate (R_A-OH) attacks the phosphoryl enzyme, forming a phosphorylated product (R_A-O-X) and returning the enzyme to the dephosphorylated state (E-O-H) (states 5, 6, and 1, respectively).

FIGURE 2.

Structural overview of B. cereus PPM. A, overall fold of B. cereus PPM. PPM is composed of a core domain and a cap domain. α-Helices are colored cyan and β-strands are colored red. The two Mn²⁺ ions are shown as a CPK representation and are colored magenta. The side chain of Thr(P)-85 is shown as a stick representation in orange. B and C, comparison of the fold of the B. cereus PPM core domain with that of E. coli alkaline phosphatase (PDB entry 1ALK) (12). The structures of PPM (B) (rotated 90° from the view in A about the black line) and alkaline phosphatase (C) (PDB entry 1ALK) (12) were aligned by their homologous metal-coordinating residues in PyMOL (26). D, topology diagram of the cap domain (residues 102–216). The color scheme is the same as in A. E, two views of electrostatic surface potential mapped onto the surface of PPM with negatively charged surfaces colored red, positively charged surfaces colored blue, and contoured from −25 to +25 kT/e, where k = Boltzmann constant, T = Temperature, and e = charge of an electron.

FIGURE 3.

Active site architecture. B. cereus PPM purifies in a mixed state, with some percentage of the protein phosphorylated and some unphosphorylated. Models depicting unphosphorylated protein use coordinates from the unphosphorylated population. A, Mn²⁺ coordination in unphosphorylated B. cereus PPM. Coordinating interactions are shown with dotted lines. B, comparison of metal coordination in B. cereus PPM (gray) and E. coli alkaline phosphatase (red) (PDB entry 1ALK) (12). Comparison was performed with a local alignment in PyMOL (26). Residues are labeled with PPM numbering first and alkaline phosphatase numbering second. C and D, occupancy of the phosphate modification of Thr-85 following incubation of B. cereus PPM with glucose 1,6-bisphosphate. 2|F_o| − |F_c| simulated annealing omit electron density maps calculated in CNS (20) after the removal of Thr-85 from the structure are contoured to 1.25 σ (green mesh). C, PPM that was not incubated with glucose 1,6-bisphosphate; D, PPM activated with glucose 1,6-bisphosphate. E, the structure of unphosphorylated B. cereus PPM (gray) overlaid with the structure of glucose 1,6-bisphosphate-activated PPM (blue). A small rotation in the χ-1 angle of Thr-85 allows the phosphate modification to bridge the two Mn²⁺ ions.

FIGURE 4.

Binding sites for substrate and activator in the B. cereus PPM active site. In A–E, electron density maps (green mesh) are 2|F_o| − |F_c| simulated annealing omit maps contoured at 1.0 σ and calculated in CNS (20) after the removal of non-protein ligands from the active site cavity. A, active site of purified PPM shows the location of water molecules within the cavity. B, active site of glucose 1,6-bisphosphate-activated PPM shows that the location of ordered water molecules within the active site does not change upon activation. C, electron density appearing in the active site of crystals of B. cereus PPM soaked with ribose 5-phosphate. D and E, interpretation of the active site density in ribose 5-phosphate soaked crystals. D, ribose 5-phosphate (R5P-1; orange sticks) bound in the coordinating position. Only electron density consistent with substrate binding at this position is shown. Putative hydrogen bonds are denoted with dotted lines. E, ribose 5-phosphate (R5P-2; orange sticks) bound in the distal position. Only electron density consistent with substrate binding at that position is shown. F, interpretation of electron density of B. cereus PPM crystals soaked with glucose 1,6-bisphosphate (G16P2; orange sticks).

FIGURE 5.

Phosphorylation of Thr-85. A, mass spectrum of the phosphorylated peptide STGKDTMTGHWEIMGL. A peptide with a mass of 1876.76 Da was isolated from the pool of peptides resulting from elastase digestion of PPM. The spectrum of the doubly charged phosphopeptide parent ion (m/z of 938.38) is shown in green. The predicted b and y ions for the phosphopeptide are shown in blue and magenta, respectively. T +80, mass of a phosphorylated threonine residue; M +16, mass of an oxidized methionine residue. B, Western analysis of phosphorylation of B. cereus PPM following incubation with glucose 1,6-bisphosphate. The upper image of Ponceau-stained nitrocellulose verifies that an equivalent amount of total protein was loaded in each lane. The lower image shows the Odyssey image of the same nitrocellulose membrane following incubation with a phosphothreonine (Thr(P))-specific primary antibody and an Alexafluor-labeled secondary antibody. The concentration of glucose 1,6-bisphosphate (in μm) is indicated above each lane. C, quantitation of data shown in B. Band intensities were calculated with the program ImageJ (30) and were normalized to the intensity of the band at 1000 μm glucose 1,6-bisphosphate in each image. The values are the average of ratios from three independent experiments; error bars represent S.D. of the mean.

FIGURE 6.

Enzymatic activity of PPM. A, the effect of glucose 1,6-bisphosphate concentration on the activity of B. cereus PPM. The calculated velocity is the average of three independent experiments. B, activity of EDTA-chelated B. cereus PPM in the presence of 1 mm cations. Activity is the average of three independent measurements, and error bars represent the S.D. of the measurement. Activity was normalized to the activity of B. cereus PPM measured after the addition of 1 mm MnCl₂. C, initial velocities of PPM as a function of ribose 5-phosphate concentration. Data are the average of three assays, and a double reciprocal plot of the data is shown in the inset. The calculated K_m, V_max, and k_cat are 263 ± 34 μm, 12.3 ± 0.7 μm ribose 5-phosphate min⁻¹, and 10.25 ± 0.6 s⁻¹.

FIGURE 7.

Isotope relay assays. A and B, comparison of phosphoryl transfer scenarios. A, schematic of intramolecular phosphoryl transfer. In intramolecular transfer, the active form of the enzyme is dephosphorylated (filled star), corresponding to Fig. 1 (state 1). B, schematic of intermolecular phosphoryl transfer. In intermolecular transfer, the active form of the enzyme is phosphorylated (filled star), corresponding to Fig. 1 (state 4). C–F, mixtures of ribose 5-phosphate synthesized from [U-¹³C₅]ribose (rings with red squares) or unenriched ribose and γ-[¹⁸O]ATP (blue) or ATP were incubated with PPM for 30 min, and the masses of the products were measured by LC-MS. C, theoretical masses for starting materials and the predicted products of either intramolecular or intermolecular phosphoryl transfer. D, mass spectra of the experiment outlined in C. In the absence of enzyme (black line) only the m/z 234 and 235 peaks corresponding to the starting materials are observed. Following incubation with PPM, peaks at m/z 229 and 240 appear (green line). The addition of human purine nucleoside phosphorylase to this reaction, which removes the product ribose 1-phosphate, decreases the intensity of these new peaks (gray line) E, theoretical masses for starting materials and the predicted products of either intramolecular or intermolecular phosphoryl transfer. F, mass spectra of the experiment outlined in E. In the absence of enzyme, only the m/z 229 and 240 peaks corresponding to the starting materials are present (black line). Incubation of the starting materials with PPM resulted in the appearance of peaks at m/z 234 and 235 (green line). These new peaks decreased in intensity following incubation with human purine nucleoside phosphorylase (gray line), which removes ribose 1-phosphate.

FIGURE 8.

Modification of the alkaline phosphatase mechanism for intermolecular phosphoryl transfer. Alkaline phosphatase follows the reaction cycle from state 1 through state 6 (red path, red substrates), starting with unphosphorylated enzyme (state 1) and phosphorylated substrate (state 2) and proceeding through a transient intermediate where the enzyme is phosphorylated (state 4) and the substrate is dephosphorylated (states 3–5) before generating a phosphorylated product (state 6). The intermolecular transfer catalyzed by PPM can occur within the context of this reaction mechanism (blue path, blue substrates) if phosphorylated enzyme (state 4) acts on phosphorylated substrate (state 5). PPM could then follow a similar path through the same catalytic cycle by proceeding through an intermediate where the enzyme is dephosphorylated (state 1) and the substrate is doubly phosphorylated (states 6, 1, and 2). To complete the reaction cycle, the bisphosphate intermediate could transfer its phosphoryl group back to the enzyme, resulting in a singly phosphorylated product (state 3) and a phosphorylated, active enzyme (state 4).