Biology, Mechanism, and Structure of Enzymes in the α-d-Phosphohexomutase Superfamily

Abstract

Enzymes in the α-d-phosphohexomutases superfamily catalyze the reversible conversion of phosphosugars, such as glucose 1-phosphate and glucose 6-phosphate. These reactions are fundamental to primary metabolism across the kingdoms of life and are required for a myriad of cellular processes, ranging from exopolysaccharide production to protein glycosylation. The subject of extensive mechanistic characterization during the latter half of the 20th century, these enzymes have recently benefitted from biophysical characterization, including X-ray crystallography, NMR, and hydrogen-deuterium exchange studies. This work has provided new insights into the unique catalytic mechanism of the superfamily, shed light on the molecular determinants of ligand recognition, and revealed the evolutionary conservation of conformational flexibility. Novel associations with inherited metabolic disease and the pathogenesis of bacterial infections have emerged, spurring renewed interest in the long-appreciated functional roles of these enzymes.

Keywords: Conformational change; Crystal structure; Enzyme mechanism; Inherited disease; Missense variants; Oligomers; Phosphorylation; Phosphosugar; Protein flexibility.

Figure 1. Overview of phosphosugar substrates and structural architecture of the PHMs

(A) Stick figures of the 1-phospho forms of various substrates of the α-D-phosphohexomutases, showing how structural differences are all localized at the 2-position of the sugar (green arrow). Also shown is a superposition of glucose 1-phosphate (white) and glucose 6-phosphate (green) illustrating the two different binding orientations (related by ~180° rotation of the sugar ring) for 1- vs. 6-phosphosugars. Superposition based on crystal structures of enzyme ligand complexes of P. aeruginosa PMM/PGM (Regni et al., 2004). (B) Schematics of the 4-domain architecture of the PHMs, highlighting the locations of key catalytic and ligand binding residues. The enzymes are typically 450-550 amino acids in length.

Figure 2. A multiple sequence alignment of one representative from each of the four major PHM sub-groups

Sequences shown are from B. anthracis PNGM, P. aeruginosa PMM/PGM, human PGM1 and C. albicans PAGM. The four (I-IV) highly conserved active site loops are highlighted (Sec. 4.3). Due to the circular permutation of the PAGM proteins relative to the others, the corresponding regions of sequence were swapped to permit the best sequence alignment (sequence between orange triangles was swapped with sequence between blue triangles). See figure key for other symbols.

Figure 3. Metabolic roles and disease-associated variants of human PGM1

(A) The pivotal position of PGM1 determining the storage of glucose as glycogen or its utilization for energy. Also shown is the connection of PGM1 to protein glycosylation, through galactose metabolism. GALT: galactose 1-phosphate uridylyltransferase; GALE: UDP-galactose-4-epimerase. (B) Ribbon diagram of human PGM1 illustrating residues affected by known missense variants (purple) associated with PGM1 deficiency. The bound metal ion in the active site is shown as an orange sphere.

Figure 4. Detailed reaction mechanism of the PHMs showing the multi-step conversion of glucose 6-phosphate to glucose 1-phosphate

The reaction proceeds via transfer of the enzymic phosphoryl group (blue) to substrate, to form a bisphosphorylated sugar intermediate. Reorientation of this intermediate (gray arrow) places the 6-phospho group (red) of the sugar near the conserved phosphoserine residue, allowing for the second phosphoryl transfer to occur back to enzyme. The product is now phosphorylated at the 1-position with the phosphoryl group (blue) derived from the enzyme. Roles for residues in acid–base catalysis are also indicated.

Figure 5. Overall structure and active site of enzymes in the major PHM sub-groups

(A) A superposition of four crystal structures, one from each major sub-group. Shown are human PGM1 (pdb: 5EPC) in pink, C. albicans PAGM (pdb: 3DKA) in blue; Bacillus anthracis PNGM (pdb: 3PDK) in green; and P. aeruginosa PMM/PGM (pdb: 1K35) in yellow. Domains 1-4 are numbered. Close-up views of the active site of (B) P. aeruginosa PMM/PGM (yellow) in complex with glucose 1-phosphate (pdb: 1P5D) and (C) C. albicans PAGM (blue) in complex with GlcNAc-1P (pdb: 2DKD). Side-by-side comparison shows the highly similar arrangements of the two active sites. Divalent metal ion is shown as gray sphere; the catalytic serine is shown in its two possible states: phosphorylated in (B) and unphosphorylated in (C). Cyan and purple stars mark putative general acid and general base, respectively. Key areas of the active site are labeled. See Fig. 2 for sequence alignments of these two proteins.

Figure 6

Structures of experimentally confirmed oligomers in the PHM superfamily. Schematics (heart shapes) and ribbon diagrams are shown for each assembly; relevant PDB IDs are noted. Domain 4 is indicated by “4” on each schematic to indicate relative orientation of the protomers within the assembly. Three different protomer-protomer interfaces are indicated by A, B, and C. Two distinct dimers and one tetramer are known. Note how domain 4 is oriented away from the assembly interfaces, permitting conformational mobility. [Reprinted from (Luebbering et al., 2012)].