Autophosphorylation of phosphoglucosamine mutase from Escherichia coli

Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-p hosphate from glucosamine-1-phosphate, an essential step in the pathway for UDP-N-acetylglucosamine biosynthesis in bacteria. This enzyme must be phos phorylated to be active and acts according to a ping-pong mechanism involvi ng glucosamine-1,6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, fur. J. Bioch em. 262:202-210, 1999). However, the process by which the initial phosphory lation of the enzyme is achieved in vivo remains unknown. Here we show that the phosphoglucosamine mutase from Escherichia coli autophosphorylates in vitro in the presence of [P-32]ATP. The same is observed with phosphoglucos amine mutases from other bacterial species, yeast N-acetylglucosamine-phosp hate mutase, and rabbit muscle phosphoglucomutase. Labeling of the E. coli GlmM enzyme with [P-32]ATP requires the presence of a divalent cation, and the label is subsequently lost when the enzyme is incubated with either of its substrates. Analysis of enzyme phosphorylation by high-pressure liquid chromatography and coupled mass spectrometry confirms that only one phospha te has been covalently linked to the enzyme. Only phosphoserine could be de tected after acid hydrolysis of the labeled protein, and site-directed muta genesis of serine residues located in or near the active site identifies th e serine residue at position 102 as the site of autophosphorylation of E. c oli GlmM.