Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

Abstract

The crystal structure of the enzyme phosphoglucomutase from Salmonella typhimurium (StPGM) is reported at 1.7 Å resolution. This is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity. A comparison of the active site of StPGM with that of other phosphoglucomutases reveals conserved residues that are likely involved in catalysis and ligand binding for the entire enzyme family. An alternate crystal form of StPGM and normal mode analysis give insights into conformational changes of the C-terminal domain that occur upon ligand binding. A novel observation from the StPGM structure is an apparent dimer in the asymmetric unit of the crystal, mediated largely through contacts in an N-terminal helix. Analytical ultracentrifugation and small-angle X-ray scattering confirm that StPGM forms a dimer in solution. Multiple sequence alignments and phylogenetic studies show that a distinct subset of bacterial PGMs share the signature dimerization helix, while other bacterial and eukaryotic PGMs are likely monomers. These structural, biochemical, and bioinformatic studies of StPGM provide insights into the large α-D-phosphohexomutase enzyme superfamily to which it belongs, and are also relevant to the design of inhibitors specific to the bacterial PGMs.

Crystal Structure of Bacillus anthracis Phosphoglucosamine Mutase, an Enzyme in the Peptidoglycan Biosynthetic Pathway

“…Also near the phosphate-binding site are two conserved threonines (residues 7 and 414 of BaPNGM) that participate in the domain 1-to-4 interface found in the closed conformer of the protein. A similar, highly conserved "threonine latch" has been observed in the crystal structures of related phosphoglucomutase enzymes (15,18).…”

“…In the closed conformer, contacts between residues in domains 1 and 4 of the protein form a lid over the active site, which is absent in the open conformer. The interdomain rotation of BaPNGM is notable for its magnitude, which is significantly larger than that seen for related proteins (typically 10 to 15°) (15,18,24). A discussion of the functional significance of the conformational variability of domain 4 can be found in this paper (see below).…”

“…Although most enzymes in the ␣-D-phosphohexomutase superfamily that have been studied to date appear to be monomeric, recent characterization of a bacterial phosphoglucomutase (PGM) from S. Typhimurium shows that this enzyme is also dimeric (15). For both BaPNGM and S. Typhimurium PGM, the dimer interface involves primarily residues in domain 1.…”

α-Fluorophosphonates reveal how a phosphomutase conserves transition state conformation over hexose recognition in its two-step reaction

“…Enzymes that operate on phosphate monoesters and anhydrides transfer the phosphoryl moiety, PO 3 − , with rate accelerations approaching 10 21 for monoesters, placing them among the most proficient of all enzymes (1). Phosphomutases, including α-phosphoglucomutase (αPGM) (2, 3) and β-phosphoglucomutase (βPGM) (4-6), phosphoglycerate mutase (7), α-phosphomannomutase (αPMM/PGM) (8), and N-acetylglucosamine-phosphate mutase (9), merit special attention because these enzymes have to be effective in donating a phosphoryl group to either of two hydroxyl groups that have intrinsically different reactivity.…”

“…Hexose 1-phosphate mutases, including enzymes central to glycolysis and other metabolic pathways, are well characterized (10,11). They are generally activated by phosphorylation to form a covalent phosphoenzyme, which then donates its PO 3 − group to either of its substrates to deliver a common, transient, hexose 1,6-bisphosphate intermediate species. However, structural studies on phosphomutases are complicated by the rapid and often imbalanced equilibrium position between the substrates, and kinetic studies are problematic because of competitive, parallel pathways of enzyme activation and substrate inhibition (12,13).…”