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KMID : 0545120100200121689
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 12 p.1689 ~ p.1695
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Identification and Characterization of a Pantothenate Kinase (PanK-sp) from Streptomyces peucetius ATCC 27952
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Ariungerel Mandakh
Narayan Prasad Niraula
Kim Eung-Pil
Sohng Jae-Kyung
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Abstract
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Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong ermE* promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.
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KEYWORD
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Coenzyme-A, Pantothenate kinase, Coupling assay, Streptomyces peucetius, Streptomyces coelicolor, Streptomyces clavuligerus
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