Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.
The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.
Monedero
V
V
Laboratoire de Génétique des Microorganismes, INRA and CNRS URA1925, Thiverval-Grignon, France.
Poncet
S
S
Mijakovic
I
I
Fieulaine
S
S
Dossonnet
V
V
Martin-Verstraete
I
I
Nessler
S
S
Deutscher
J
J
D000483:Alleles; D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins; D050260:Carbohydrate Metabolism; D002244:Carbon / Q000378:metabolism*; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D007763:Lac Operon; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D002244:Carbon; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases