An eukaryotic-type serine/threonine protein kinase involved in the carbon source-dependent pigment biosynthesis in Amycolatopsis mediterranei U32.
The structural gene, pkmA, was cloned and sequenced from a rifamycin SV-producing Amycolatopsis mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the family of serine/threonine protein kinases. It contains all the structural features which are highly conserved in protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein specific kinases. The protein possesses a region rich in Ala and Pro residues around the middle of pkmA open reading frame, which might be involved in the transmembrane function, as suggested by PhoA fusion protein analysis. The pkmA gene was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, and the protein was found to have the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. mediterranei chromosomal DNA. The phenotypic analysis of the mutant suggested that pkmA gene is involved in carbon source-dependent pigment formation in A. mediterranei U32.
Yang
L
L
Laboratory of Molecular Microbiology, Shanghai Institutes for Biological Sciences, Shanghai, 200032, People's Republic of China.
Zhang
W
W
Chiao
J
J
Zhao
G
G
Jiang
W
W
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009615:Nocardia / Q000201:enzymology* / Q000254:growth & development; D010641:Phenotype; D010860:Pigments, Biological / Q000032:analysis / Q000096:biosynthesis*; D011134:Polysaccharides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012294:Rifamycins / Q000096:biosynthesis; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry
D001426:Bacterial Proteins; D010860:Pigments, Biological; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D012294:Rifamycins; C023808:rifamycin SV; C430089:PkmA protein, Amycolatopsis mediterranei; D017346:Protein Serine-Threonine Kinases; D000469:Alkaline Phosphatase