Purification and enzymatic characterization of PgcM: a β-phosphoglucomutase and glucose-1-phosphate phosphodismutase of Bacillus subtilis

Abstract.

Phosphoglucomutases catalyze the reversible conversion of D-glucose 1-phosphate to D-glucose 6-phosphate, a key metabolic step in all cells. Two classes of phosphoglucomutases have been described so far, using either the α- or β-forms of the phosphorylated sugars. The pgcM gene of Bacillus subtilis was cloned and used to construct a plasmid-based overexpression system for PgcM in Bacillus megaterium. The obtained protein was purified and its enzymatic activities were characterized. PgcM exhibits β-phosphoglucomutase activity, transforming mainly β-glucose 1-phosphate to β-glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Nevertheless, α-glucose 1-phosphate can also serve as a substrate, but with a seven-fold lower affinity than that observed for the β-form. Additionally, PgcM exhibits a glucose-1-phosphate phosphodismutase activity using the α- and β-forms as substrates, with affinities comparable to those observed for the phosphoglucomutase activity. Conformational changes of PgcM triggered by cofactors (MgCl₂, glucose 1,6-bisphosphate) and substrate (glucose 1-phosphate) were detected by fluorescence spectra. Insertional mutagenesis of pgcM resulted in an inactivation of β-phosphoglucomutase activity in B. subtilis. These mutants showed growth deficiency on minimal medium containing starch or maltodextrins (maltose to maltoheptaose) compared either to the wild-type or to growth on minimal medium containing glucose.