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

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

doi:10.1074/jbc.272.22.14166

Abstract

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.

Highlights

Glycerol uptake in Gram-negative and Gram-positive bacteria is mediated by the glycerol diffusion facilitator, an integral membrane protein of 30 kDa, catalyzing the rapid equilibration of concentration gradients of glycerol across the cytoplasmic membrane [1, 2]
Partial sequence analysis after subcloning into pCR-Script confirmed the presence of both glpK and glpO coding sequences, and the size of the product is consistent with that predicted from the B. subtilis glpKD sequence and the peptide alignments corresponding to glk1 and gpo3
This glpKO polymerase chain reaction (PCR) product was mapped in pCR-Script with a series of restriction digests [25]; both the 1.1-kb ClaI fragment corresponding to glpK and the 2.4-kb PCR product were used as probes in the glpK cloning described

Summary

EXPERIMENTAL PROCEDURES

Materials—Isopropyl 1-thio-␤-D-galactopyranoside (IPTG) was purchased from 5Prime-3Prime, Inc., or from TEBU, France, and agarose was from FMC Bioproducts. ␥-[32P]ATP was purchased from Amersham, France. A Southern blot of E. faecalis genomic DNA was probed with a digoxigenin-labeled DNA fragment containing the entire E. casseliflavus glpK gene, to identify a 1.6-kb EcoRI/BamHI fragment for cloning Sequence analysis of this EcoRI/BamHI clone in pMOB (pGLP12) indicated that it lacked approximately 150 –180 bp corresponding to the 5Ј-end of the gene. The PCR-generated fragment of glpK was sequenced to confirm the subcloning This plasmid was transformed into E. coli BL21DE3 for expression of the E. faecalis glycerol kinase after induction with IPTG, which gave better results than the strain JM109DE3 that we used for expression of glpK from E. casseliflavus. The instrument was calibrated using human insulin and horse hemoglobin as standards

RESULTS

DISCUSSION

Enzyme activityb