Autophosphorylation of phosphoglucosamine mutase from Escherichia coli.
Phosphoglucosamine mutase (GlmM) catalyzes the formation of glucosamine-1-phosphate from glucosamine-6-phosphate, an essential step in the pathway for UDP-N-acetylglucosamine biosynthesis in bacteria. This enzyme must be phosphorylated to be active and acts according to a ping-pong mechanism involving glucosamine-1, 6-diphosphate as an intermediate (L. Jolly, P. Ferrari, D. Blanot, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx, Eur. J. Biochem. 262:202-210, 1999). However, the process by which the initial phosphorylation 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 [(32)P]ATP. The same is observed with phosphoglucosamine mutases from other bacterial species, yeast N-acetylglucosamine-phosphate mutase, and rabbit muscle phosphoglucomutase. Labeling of the E. coli GlmM enzyme with [(32)P]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 phosphate has been covalently linked to the enzyme. Only phosphoserine could be detected after acid hydrolysis of the labeled protein, and site-directed mutagenesis of serine residues located in or near the active site identifies the serine residue at position 102 as the site of autophosphorylation of E. coli GlmM.
Jolly
L
L
Laboratoire des Enveloppes Bactériennes et Antibiotiques, Centre National de la Recherche Scientifique, Université Paris-Sud, 91405 Orsay, France.
Pompeo
F
F
van Heijenoort
J
J
Fassy
F
F
Mengin-Lecreulx
D
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D013058:Mass Spectrometry; D010733:Phosphoglucomutase / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation / Q000187:drug effects
D010761:Phosphorus Radioisotopes; D015636:Magnesium Chloride; D000255:Adenosine Triphosphate; D004492:Edetic Acid; C097631:phosphoglucosamine mutase; D010733:Phosphoglucomutase