Probing the role of cysteine residues in glucosamine-1-phosphate acetyltransferase activity of the bifunctional GlmU protein from Escherichia coli: site-directed mutagenesis and characterization of the mutant enzymes.
The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from Escherichia coli was lost when GlmU was stored in the absence of beta-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The Km values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.
Pompeo
F
F
Biochimie Structurale et Cellulaire, Centre National de la Recherche Scientifique, Université Paris-Sud, 91405 Orsay Cedex, France.
van Heijenoort
J
J
Mengin-Lecreulx
D
D
D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine; D005944:Glucosamine / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D005958:Glucosephosphates; D000123:Acetyltransferases; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D003545:Cysteine; D005944:Glucosamine