Cloning and Characterization of the HPr Kinase/Phosphorylase Gene from Bacillus stearothermophilus No. 236

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概要

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• 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.

• 社団法人 日本農芸化学会の論文
• 2006-05-23

著者

• Choi Y‐j

  Korea Inst. Sci. And Technol. Seoul Kor

• Choi Y‐j

  Korea Univ. Seoul Kor

• Choi Ildong

  Graduate School Of Life Sciences And Biotechnology Korea University

• Kim Kyung-nam

  Molecular Biology Sejong University

• Yun Cheol-won

  Graduate School Of Life Sciences And Biotechnology Korea University

• CHOI Il-Dong

  Graduate School of Life Sciences and Biotechnology, Korea University

• CHOI Yong-Jin

  Graduate School of Life Sciences and Biotechnology, Korea University

• Choi Yong-Jin

  Graduate School of Biotechnology, Korea University

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• Cloning and Characterization of the HPr Kinase/Phosphorylase Gene from Bacillus stearothermophilus No. 236
• Cloning and Characterization of the Catabolite Control Protein A Gene from Bacillus stearothermophilus No. 236
• Catabolite Repression of the Xylanase Gene (xynA) Expression in Bacillus stearothermophilus No.236 and B.subtilis
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