Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect

Abstract

Phosphomannomutase/phosphoglucomutase contributes to infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. NMR spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. 13C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These are attributable to the phosphorylation state and possibly to conformational interconversions. S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose-1-phosphate substrate by impairment of kcat. Addition of the glucose-1,6-bisphosphate intermediate accelerated this reaction by 2 – 3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft, while preserving the secondary structure in solution. Diminished peak heights and faster 15N relaxation are suggestive of line broadening and millisecond fluctuations within four loops that can contact phosphosugars. 15N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1 to 3, and with a different principal axis of diffusion. This adds to the crystallographic evidence for domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of intermediate, substrate binding, and product release.

Promotion of Enzyme Flexibility by Dephosphorylation and Coupling to the Catalytic Mechanism of a Phosphohexomutase

“…The existence of multiple conformers for PMM/PGM is supported by crystal structures ( Fig. 2A) (7,8,26) and by NMR studies showing that D4 has higher relative mobility in solution than the rest of the protein (14). A comprehensive view of peptides affected by phosphorylation throughout the time course (not just at the final time point as in Fig.…”

“…S3A). These are among several loops with internal motions suggested by NMR (14), hinting at a role for phosphorylation in modifying the conformational flexibility of loops within the active site cleft. We note that a number of the affected peptides are Ͼ10 Å away from the site of phosphorylation and outside of the active site (e.g.…”

“…Some of these effects can be rationalized by previous analyses of the D4 conformational change. Using available crystal structures of PMM/PGM, both principal component and normal mode analyses (11,14) suggest that the rotation of D4 occurs via a hinge type motion. Therefore, it is simple to envision that the rigid body motion of D4 that produces the closed, ligand-binding conformer of the protein can create a more open conformer as well.…”

“…Protein Preparation-Expression and purification of P. aeruginosa PMM/PGM and its phosphorylation/dephosphorylation were performed as described previously (13,14). Briefly, phosphorylation and dephosphorylation were achieved by incubating the protein with glucose 1,6-bisphosphate or glucosamine 1-phosphate, respectively, at 6-fold molar excess over enzyme at 4°C for 18 h. Ligands were removed by extensive dialysis against 50 mM MOPs, pH 7.4 and 1 mM MgCl 2 .…”

Conservation of Functionally Important Global Motions in an Enzyme Superfamily across Varying Quaternary Structures

“…Both Oryctolagus cuniculus PGM (OcPGM) and Pseudomonas aeruginosa PMM/PGM (PaPMM) have been the subject of extensive biochemical and kinetic studies. 11,12,15,17,19,[29][30][31][32][33][34] The PAGM and PNGM proteins of known structure have clear sequence homology to functionally characterized homologs. 35 However, based on sequence comparisons, we find two proteins in Table 1 difficult to place in a specific subgroup of the superfamily and therefore designate them as "ambiguous".…”