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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 hprK V267F 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.