Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
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Molecular Cloning and Analysis of the Gene for P-450 Hydroxylase from Pseudonocardia autotrophica IFO 12743

  • Kim, Jung-Mee (Department of Biological Science, Myong Ji University, Atman Bioscience Inc., Wanggok-Dong, Uiwang City) ;
  • Younmie Jin (Department of Biological Science, Myong Ji University) ;
  • Hyun, Chang-Gu (Department of Biological Science, Myong Ji University, Atman Bioscience Inc., Wanggok-Dong, Uiwang City) ;
  • Kim, Jong-Hee (Department of Biological Science, Myong Ji University) ;
  • Lee, Hong-Sub (Ildong Pharmaceutical Co., LTD.) ;
  • Kang, Dae-Kyung (Bio-Resources Institute, Easy Bio System Inc.) ;
  • Kang, Dae-Jung (Ildong Pharmaceutical Co., LTD.) ;
  • Kim, Tae-Yong (Ildong Pharmaceutical Co., LTD.) ;
  • Suh, Joo-Won (Department of Biological Science, Myong Ji University)
  • Published : 2002.09.01
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Abstract

A 4.8-kb DNA fragment encoding the P-450 type hydroxylase and ferredoxin genes was cloned from Pseudonocardia autotrophica IFO 12743 that can convert vitamin D$\_$3/ into its hydroxylated active forms. In order to isolate the P-450 gene cluster in this organism, we designed PCR primers on the basis of the regions of an oxygen binding site and a heme ligand pocket that are general characteristics of the P-450 hydroxylase. Sequencing analysis of the BamHI fragment revealed the presence of four complete and one incomplete ORFs, named PauA, PauB, PauC, and PauD, respectively. As a result of computer-based analyses, PauA and PauB have homology with enoyl-CoA hydratase from several organisms and the positive regulators belonging to the tetR family, respectively. PauC and PauD show similarity with SuaB/C proteins and ferredoxins, respectively, which are composed of P-450 monooxygenase systems for metabolizing two sulfonylurea herbicides in Streptomyces griseolus PauC shows the highest similarity with another CytP-450$\_$Sca2/ protein that is responsible for production of a specific HMG-CoA reductase inhibitor, pravastatin, in S. carbophilus. Cultures of Steptomyces lividans transformant, containing the P-450 gene cluster on the pWHM3 plasmid, was unable to convert vitamin D$\_$3/ to its hydroxylated forms.

Keywords

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