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

Il Dong Choi, Kyung Nam Kim, Cheol Won Yun, Yong Jin Choi

Research output: Contribution to journalArticle

3 Citations (Scopus)

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°C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45°C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40°C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg2+, 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.

Original languageEnglish
Pages (from-to)1089-1101
Number of pages13
JournalBioscience, Biotechnology and Biochemistry
Volume70
Issue number5
DOIs
Publication statusPublished - 2006

Keywords

  • Bacillus stearothermophilus
  • Carbon catabolite repression
  • Catabolite control protein A
  • HPr kinase/phosphorylase

ASJC Scopus subject areas

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

Fingerprint Dive into the research topics of 'Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus no. 236'. Together they form a unique fingerprint.

Cite this