Cloning and sequencing of two enterococcal glpK genes and regulation of the encoded glycerol kinases by phosphoenolpyruvate-dependent, phosphotransferase system-catalyzed phosphorylation of a single histidyl residue.
The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. The translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. After expression of the enterococcal glpK genes in Escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme I and the histidine-containing protein of the phosphoenolpyruvate:glycose phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fold increase in enzyme activity. The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis was used to replace His-232 in glycerol kinase of E. casseliflavus with an alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be phosphorylated confirming that His-232 of E. casseliflavus glycerol kinase represents the site of phosphorylation. The His232 --> Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavus glycerol kinase activity. However, neither EIIAGlc from E. coli nor the EIIAGlc domain of Bacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.
Charrier
V
V
Institut de Biologie et Chimie des Protéines, CNRS, 7 passage du Vercors, F-69367 Lyon Cedex 07, France.
Buckley
E
E
Parsonage
D
D
Galinier
A
A
Darbon
E
E
Jaquinod
M
M
Forest
E
E
Deutscher
J
J
Claiborne
A
A
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016983:Enterococcus / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005991:Glycerol Kinase / Q000235:genetics*; D006639:Histidine / Q000378:metabolism; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013329:Structure-Activity Relationship
D006639:Histidine; D010728:Phosphoenolpyruvate; D010770:Phosphotransferases; D005991:Glycerol Kinase