Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236.
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.
Choi
Il-Dong
ID
Graduate School of Life Sciences and Biotechnology, Korea University, Seoul.
Kim
Kyung-Nam
KN
Yun
Cheol-Won
CW
Choi
Yong-Jin
YJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular; D005635:Fructosediphosphates / Q000737:chemistry; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010710:Phosphates / Q000737:chemistry; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein
D001426:Bacterial Proteins; D002413:Cations, Divalent; D005635:Fructosediphosphates; D010710:Phosphates; D006005:Phosphorylases; C110847:HPr kinase; D017346:Protein Serine-Threonine Kinases; C029063:fructose-1,6-diphosphate