Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase.
We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169 degrees on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3' part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identify. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.
Fischer
C
C
Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Geourjon
C
C
Bourson
C
C
Deutscher
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008353:Mannitol / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017421:Sequence Analysis; D012694:Serine / Q000378:metabolism; D014170:Transformation, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D008353:Mannitol; D012694:Serine; D011494:Protein Kinases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; C098604:PrkA protein, Bacillus subtilis; D017346:Protein Serine-Threonine Kinases; D008274:Magnesium