HPr kinase/phosphatase of Bacillus subtilis: expression of the gene and effects of mutations on enzyme activity, growth and carbon catabolite repression

Abstract

HPr kinase/phosphatase (HPrK/P) is the key protein in regulation of carbon metabolism in Bacillus subtilis and many other Gram-positive bacteria. Whether this enzyme acts as a kinase or phosphatase is determined by the nutrient status of the cell. Mutational analysis of residues in a Walker A box nucleotide-binding motif revealed that it is not only important for kinase but is also involved in phosphatase activity. In addition, a signature sequence specifically conserved among HPrK/P orthologues is required for phosphatase activity and may be involved in interaction with HPr/HPr-(Ser46)-P. Carbon catabolite repression was abolished in a B. subtilis strain expressing a mutant form of HPrK/P deficient in kinase and phosphatase activities. The growth characteristics of this strain were similar to those of the wild-type. In contrast, B. subtilis strains expressing HPrK/P with partial kinase and no phosphatase activities showed growth impairment but exhibited catabolite repression.

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…Such mutants have been obtained, for example, with B. subtilis (251,315,709), S. xylosus (357), and L. casei (198). In all three organisms, the inactivation of hprK causes a relief from CCR, but for several B. subtilis catabolic operons, the releasing effect in hprK mutants is markedly stronger than that in ptsH1 mutants (249,523,685).…”

“…The inhibitory effect of ATP was stronger at low Mg 2ϩ concentrations. Mutant studies with hprK from various organisms suggested that Walker motif A is important not only for the kinase but also for the phosphorylase function (315,571,831).…”

Catabolite Repression and Activation in Bacillus subtilis : Dependency on CcpA, HPr, and HprK

“…(x) Effects of hprK mutations alter growth independently of their effects on CR (22). (xi) Several reports have demonstrated the existence of CcpA-independent CR in B. subtilis (7,8,64,73,78,80).…”

HPr Kinase/Phosphorylase, the Sensor Enzyme of Catabolite Repression in Gram-Positive Bacteria: Structural Aspects of the Enzyme and the Complex with Its Protein Substrate

“…By contrast, Lys160Ala and Lys160Arg substitutions in M. pneumoniae HprK/P led to a complete loss of the two activities (47). Several other mutations affecting amino acids in the P-loop of B. subtilis HprK/P reduced or abolished both catalytic activities (21). The replacement of Gly160 with a serine in L. casei HprK/P, while reducing the phosphorylase activity, had little effect on the kinase activity (38) and yielded a phenotype similar to that observed for mutations affecting the central loop.…”

“…The so-called capping motif (15), an unusual structure corresponding to the conserved HprK/P signature sequence (43), completes the globular core of the HprK/P subunit. Mutations affecting amino acids in the signature sequence of HprK/P from M. pneumoniae and B. subtilis abolished the dephosphorylation activity, and some also reduced the phosphorylation activity (21,47).…”