Cloning and Characterization of the HPr Kinase/Phosphorylase Gene from Bacillus stearothermophilus No. 236
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The <I>Bacillus stearothermophilus</I> 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)<SUB>6</SUB>-tagged gene product was characterized in detail. The nucleotide sequence of the <I>hprK/P</I> 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 <I>B. stearothermophilus</I> no. 236 HprK/P showed 64.5% identity with the <I>B. subtilis</I> 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 <I>hprK/P</I> gene product was functionally active in the <I>B. subtilis</I> cells. The purified (His)<SUB>6</SUB>-tagged <I>B. stearothermophilus</I> 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 Mg<SUP>2+</SUP>, 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.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(5), 1089-1101, 2006-05-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry