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-pro ducing Amycolatopsis mediterranei U32 strain. The N-terminal portion of the deduced amino acid sequence of pkmA showed significant similarity to the f amily of serine/threonine protein kinases. It contains all the structural f eatures 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/th reonine protein specific kinases. The protein possesses a region rich in Al a and Pro residues around the middle of pkmA open reading frame, which migh t be involved in the transmembrane function, as suggested by PhoA fusion pr otein analysis. The pkmA gene was expressed in Escherichia coil as a glutat hione S-transferase (G;ST) fusion protein, and the protein was found to hav e the activity of autophosphorylation. A double crossover gene replacement was achieved by inserting an aparmycin resistance gene into pkmA in A. medi terranei chromosomal DNA. The phenotypic analysis of the mutant suggested t hat pkmA gene is involved in carbon source-dependent pigment formation in A . mediterranei U32. (C) 2001 Academic Press.