Purification and enzymatic characterization of PgcM: a beta-phosphoglucomutase and glucose-1-phosphate phosphodismutase of Bacillus subtilis.
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 alpha- or beta-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 beta-phosphoglucomutase activity, transforming mainly beta-glucose 1-phosphate to beta-glucose 6-phosphate via the intermediate glucose 1,6-bisphosphate. Nevertheless, alpha-glucose 1-phosphate can also serve as a substrate, but with a seven-fold lower affinity than that observed for the beta-form. Additionally, PgcM exhibits a glucose-1-phosphate phosphodismutase activity using the alpha- and beta-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 beta-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.
Mesak
L R
LR
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik, Universität Erlangen-Nürnberg, Germany.
Dahl
M K
MK
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D003470:Culture Media; D007700:Kinetics; D009154:Mutation; D010733:Phosphoglucomutase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D003470:Culture Media; D010770:Phosphotransferases; C046332:glucose-1-phosphate phosphodismutase; D010733:Phosphoglucomutase