Phosphorylation-Dependent Effects on the Structural Flexibility of Phosphoglucosamine Mutase from Bacillus anthracis

Abstract

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. 15 N 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.

Structural and dynamical description of the enzymatic reaction of a phosphohexomutase

“…Also as part of this study, we characterized a series of “off pathway” complexes of XcPGM. These include E deP with a bound substrate or product (states 8–11 on Table I) as well as an E P :I complex (state 14 ) also previously determined 3 . None of these are part of the productive catalytic cycle, but may occur, for example, if the enzyme binds the ligand after losing its phosphoryl group from hydrolysis.…”

“…1(a)]. As noted above, hydrogen-deuterium exchange by mass spectrometry, NMR, and various biochemical studies of other enzymes in the superfamily has suggested that E deP has increased flexibility of its polypeptide backbone relative to E P. 8,9,11,18 These flexibility changes, which have not been apparent in crystal structures, have been proposed to facilitate the release and reorientation of G16P 9,11 …”

“…Comparisons of the polypeptide backbone in the various XcPGM structures reveal conformational changes in two active site loops, related in one case to ligand-binding, loop (iv), and, in the other, to a change in the phosphorylation state of the catalytic serine in loop (i). While the former has been seen in other enzymes in the superfamily, 14,37 direct structural changes related to phosphorylation have not been characterized previously, although other effects of this covalent modification have been noted 8,10,11,18 . Thus the XcPGM structures add to the types of conformational variations associated with the catalytic cycle of the α-D-phosphohexomutases.…”

“…Adding to the complexity of recognition, certain subgroups of the superfamily have dual substrate specificity, such as the PMM/PGMs that can effectively utilize both glucose and mannose-based substrates 7 . Moreover, detailed biophysical studies of several enzymes in the superfamily have established that loss of covalent modification of the catalytic serine by phosphorylation increases the flexibility of the polypeptide backbone, 8–11 a phenomenon that may facilitate the release and rebinding of the bisphosphorylated sugar intermediate during the course of the reaction 9 . Overall, the catalytic mechanism of these enzymes demands a robustly designed active site that can accommodate different ligand binding orientations, recognize varying types of sugars and number of phosphorylation sites, and enable the reorientation of the intermediate in the midst of the catalytic cycle.…”

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

“…In other members of the superfamily, similar biochemical evidence is mounting for phosphorylation- dependent differences in protein flexibility (Y. Lee, Stiers, et al, 2014; Stiers, Xu, Lee, & Beamer, n.d.), suggesting that this is a conserved feature of the PHMs, and supporting its functional importance in enzyme mechanism.…”