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Volume 272, Number 22, Issue of May 30, 1997 pp. 14166-14174
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, February 18, 1997, and in revised form, March 24, 1997)

Véronique Charrier , Ellen Buckley ^§ , Derek Parsonage ^§ , Anne Galinier , Emmanuelle Darbon , Michel Jaquinod ^¶ , Eric Forest ^¶ , Josef Deutscher and Al Claiborne ^§

From the  Institut de Biologie et Chimie des Protéines, CNRS, 7 passage du Vercors, F-69367 Lyon Cedex 07, France, ^§ Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, North Carolina 27157, and ^¶ Institut de Biologie Structurale, CNRS, 41 avenue des Martyrs, F-38027 Grenoble Cedex 1, France

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 EIIA^Glc from E. coli nor the EIIA^Glc domain of Bacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.

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