Cloning, sequencing and expression of serine/threonine kinase-encoding genes from Streptomyces coelicolor A3(2).
A 6.3-kb DNA fragment encoding two eukaryotic-type serine/threonine protein kinases (Ser/Thr PK) was cloned from Streptomyces coelicolor A3(2) by using a PCR product obtained with primers based on highly conserved regions of eukaryotic Ser/Thr PK. The nucleotide (nt) sequence of the essential 4.4-kb fragment contained two possible ORFs. One ORF (PkaA) contained 543 amino acids (aa), while another (PkaB) consisted of 417 aa. The N-terminal half of both proteins showed significant similarity with the catalytic domain of eukaryotic Ser/Thr PK. On the other hand, the C-terminal region of PkaA, but not of PkaB, is rich in Pro and Gln residues, indicating that PkaA works as a PK as well as a transcription factor. The pkaB gene was overexpressed in Escherichia coli, and the gene product (PkaB) was found to be phosphorylated mainly at Thr. The pkaA gene was also overexpressed in E. coli, and the gene product (PkaA) was found to be phosphorylated mainly at Thr and slightly at Ser. In the case of PkaA, at least 100 aa residues from the C terminus were not essential for the PK activity. When the PCR product was used as a probe, it hybridized to DNA fragments from all the Streptomyces species tested, indicating that these types of Ser/Thr PK are distributed ubiquitously and play significant physiological roles in the various species of Streptomyces.
Urabe
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Department of Biochemistry, Meiji College of Pharmacy, Tokyo, Japan.
Ogawara
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D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000201:enzymology; D005798:Genes, Bacterial; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D014157:Transcription Factors
D001426:Bacterial Proteins; D014157:Transcription Factors; C092410:PkaA protein, Streptomyces coelicolor; C092411:PkaB protein, Streptomyces coelicolor; D017346:Protein Serine-Threonine Kinases