RESUMEN
Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme in the peptidoglycan biosynthetic pathway, catalyzing the reversible conversion between glucosamine 1- and 6-phosphate. Previous structural studies of PNGM from the pathogen Bacillus anthracis revealed its dimeric assembly and highlighted the rotational mobility of its C-terminal domain. Recent studies of two other enzymes in the same superfamily have demonstrated the long-range effects on the conformational flexibility associated with phosphorylation of the conserved, active site phosphoserine involved in phosphoryl transfer. Building on this work, we use a combination of experimental and computational studies to show that the active, phosphorylated version of B. anthracis PNGM has decreased flexibility relative to its inactive, dephosphorylated state. Limited proteolysis reveals an enhanced and accelerated cleavage of the dephosphorylated enzyme. 15N transverse relaxation-optimized NMR spectra corroborate a conformational adjustment with broadening and shifts of peaks relative to the phospho-enzyme. Electrostatic calculations indicate that residues in the mobile, C-terminal domain are linked to the phosphoserine by lines of attraction that are absent in the dephosphorylated enzyme. Phosphorylation-dependent changes in protein flexibility appear linked with the conformational change and enzyme mechanism in PNGM, establishing this as a conserved theme in multiple subgroups of the diverse α-d-phosphohexomutase superfamily.