Promotion of enzyme flexibility by dephosphorylation and coupling to the catalytic mechanism of a phosphohexomutase

Abstract

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.

Keywords: Carbohydrate Biosynthesis; Enzyme Catalysis; Hydrogen-Deuterium Exchange; Phosphorylation; Protein Dynamics; Pseudomonas aeruginosa; Small Angle X-ray Scattering.

FIGURE 1.

Reaction of the α-d-phosphohexomutases. The reaction is illustrated for P. aeruginosa PMM/PGM with glucose 1-phosphate as substrate. The two phosphoryl groups are shown as circles with either black or white backgrounds to highlight their respective transfers to/from enzyme. The required reorientation of glucose 1,6-bisphosphate is indicated by a gray arrow.

FIGURE 2.

Structure of P. aeruginosa PMM/PGM. A, a superposition of two crystal structures (Protein Data Bank codes 1P5D and 1K2Y) showing conformational variability between the apoenzyme and ligand-bound enzyme. The four structural domains (D1–D4) of the protein are in yellow, green, light blue, and pink, respectively. B, a close-up view of the active site in the enzyme-ligand complex (Protein Data Bank code 1P5D) with glucose 1-phosphate (G1P). Phosphoserine 108 and the side chains of other ligand-binding residues are shown as sticks; the metal ion is a purple sphere. The color scheme is the same as that in A.

FIGURE 3.

Comparison of HDXMS data from PMM/PGM samples. A, histogram summarizing the change in deuterium incorporation over time. Peptides from the pepsin digest are listed from the N to C terminus (x axis). The relative percentage of deuterium (deuterium exchanged/maximum exchangeable amides × 100) is on the y axis. Above each peptide is a triplet of bars corresponding to the apo-deP, apo-P, and X1P-P samples, respectively. Each bar is colored in segments corresponding to the time point of analysis. Structural domains (D1–D4) are shown in the inset; red arrows indicates the location of the catalytic phosphoserine residue. Numbers in circles (1–5) show peptides with a slightly different residue range than on that on the x axis: 53–72, 157–168, 329–349, 350–370, and 432–441, respectively. B, a butterfly plot comparing the apo-P (top) and apo-deP (bottom) samples of PMM/PGM at five selected time points. Axes are the same as those in A. Each trace represents a time point of exchange (see box for color scheme); discontinuities reflect missing data for several peptides. Striped vertical bars highlight peptides that differ by >30% in deuterium content at the first (pink) or final time point (blue).

FIGURE 4.

Differential deuterium incorporation on structure of PMM/PGM and in peptide time courses. A, difference in percentage of relative deuterium content shown by peptide between apo-deP versus apo-P (left) and apo-P versus X1P-P (right) samples at the final time point of ∼1200 min. Color indicates a difference: red, >30%; orange, <30%; yellow, <20%; blue, <10%; gray indicates missing peptide. Peptides with spheres have the greatest difference between sample pairs: left, peptides 267–293, 407–429, and 442–463; right, peptides 294–303, 378–406, and 432–441. The green arrow in the left panel highlights phosphoserine residue. B, time courses of deuterium incorporation for selected peptides from A with contrasting rates and level of incorporation between samples: apo-deP, black circles; apo-P, red circles; X1P-P, blue circles. The maximum value on the y axis corresponds to the total possible number of exchangeable deuteriums. Boxed numbers show location of peptides in A and their corresponding time courses in B.

FIGURE 5.

SAXS data. Solution properties derived from SAXS for three forms of PMM/PGM, apo-deP (black), apo-P (red), and X1P-P (blue), are shown. A, normalized pair distribution function P(r) plot showing broadening of apo-deP relative to the others. B, Porod-Debye plot showing plateaus indicative of well folded globular proteins with differences in structural compactness. C, superposition of the phosphoenzyme (Protein Data Bank code 1K35) and dephosphoenzyme (Protein Data Bank code 4MRQ; this study) crystal structures of PMM/PGM. The apo-P enzyme is shown in orange; the apo-deP structure is shown in gray.