Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236

Biosci Biotechnol Biochem. 2006 May;70(5):1089-101. doi: 10.1271/bbb.70.1089.

Abstract

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Base Sequence
  • Cations, Divalent / chemistry
  • Cloning, Molecular
  • Fructosediphosphates / chemistry
  • Geobacillus stearothermophilus / enzymology*
  • Geobacillus stearothermophilus / genetics
  • Hydrogen-Ion Concentration
  • Molecular Sequence Data
  • Molecular Weight
  • Phosphates / chemistry
  • Phosphorylases / chemistry
  • Phosphorylases / genetics
  • Phosphorylases / isolation & purification
  • Protein Serine-Threonine Kinases / chemistry*
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / isolation & purification
  • Sequence Analysis, Protein

Substances

  • Bacterial Proteins
  • Cations, Divalent
  • Fructosediphosphates
  • Phosphates
  • Phosphorylases
  • HPr kinase
  • Protein Serine-Threonine Kinases
  • fructose-1,6-diphosphate