The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

摘要: Zeneca Pharmaceuticals, Mereside, Alderley Park,Macclesfield, Cheshire SK10 4TG, UK.SummaryThe HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphory-latesthe HPrprotein the bacterial phosphotransfer-ase system (PTS) and involved in regulation ofcarbohydratemetabolism. The hprKgene from Entero-coccus faecaliswas cloned via polymerase chain reac-tion (PCR) sequenced. deduced amino acidsequencewasconfirmedbymicroscaleEdmandegra-dation mass spectrometry combined with colli-sion-induced dissociation tryptic peptides derivedfrom E. faecalis. gene was over-expressedin Escherichia coli, doesnot containany or phosphatase activ-ity. homogeneous recombinant exhibitsthe expected activity as well a P-Ser-HPr activity, assumed to be aseparate enzyme activity. bifunctional kinase/phosphatase acts preferentially at highATP levels 2mM occurring glucose-metabolizingStreptococci. At low ATP levels, hydro-lyses P-Ser-HPr. In addition, high concentrations ofphosphate present under starvation conditions inhibitthe Thus, putative function theenzymemaybetoadjustthe ratio ofHPrand P-Ser-HPraccording metabolic state cell; P-Ser-HPris carbon catabolite repression regu-latessugaruptakeviathephosphotransferasesystem(PTS). Reinvestigation previously describedBacillus subtilisHPr revealed that it also pos-sesses However, con-trary faecalis enzyme, alone notsufficient switch B.subtilis A change inactivity oftheB.subtilisHPrkinase onlyobservedwhen fructose-1,6-bisphosphate present.IntroductionATP-dependent kinases are known play keyrole metabolism signal transduc-tion eukaryotic organisms phosphorylating seryl, threo-nylandtyrosylresiduesina distinctstructuralenvironment(Hunter, 1991). bacteria, only very few ATP-dependentprotein so far. One extensively studiedsystem isthe isocitratedehydrogenase kinase,whichphos-phorylates isocitrate dehydrogenase (IDH) single serylresidue(Ser-113),leadingtotheinactivationoftheIDH(LaPorte, 1993).This regulatory mechanismenablesthe bac-terial cell bypass citric acid cycle glyoxylatepathway. phosphorylation/dephosphorylation IDHis catalysed by called IDHkinase/phosphatase (Rittinger et al., 1996).A second highly specific kinasewas discovered Streptococcus pyogenes (Deutscherand Saier, 1983) partially purified fromEnterococcusfaecalis(DeutscherandEngelmann,1984).Inthepresenceof glucose, this phosphorylates phosphocarrierprotein PEP-dependent phosphotransferasesystem (PTS; reviewed Postma 1993) Ser-46(Deutscher 1986). During phos-photransfer reaction, phosphorylated His-15 byenzyme I. phosphorylationdrastically inhibits phosphorylation ofHPr (Deutscheret 1985). As result, thesugar uptake PTS almost abolished. additionto inhibitionof involvedin expression various catabolic genesin bacteria. has been shownto bind specifically CcpA transcription factor.