Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase |
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Affiliation: | 1. Department of Chemistry, Biology, and Environmental Sciences, Nara Women''s University, Kita-Uoya Nishimachi, Nara 630-8506, Japan;2. Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, Gakuen 2-1, Sanda-shi, Hyogo 669-1337, Japan;2. Department of Chemistry, University of Wisconsin–Whitewater, Whitewater, Wisconsin, USA;3. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA;4. Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA;5. The Eppley Institute and the Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA |
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Abstract: | We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° 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) (25 038 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 identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 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. |
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