Purification and characterization of the 3-ketosteroid-δ-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans

Kwang Pil Choi, István Molnár, Mitsuo Yamashita, Yoshikatsu Murooka

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The 3-ketosteroid-Δ1-dehydrogenase (KS1DH) gene of Arthrobacter simplex IFO12069 cloned in Streptomyces lividans was overexpressed, resulting in production of the enzyme both extracellularly and intracellularly. The enzyme was purified by ammonium sulfate fractionation and chromatographies using DEAE-Toyopearl, Butyl-Toyopearl and Toyo-pearl HW55S from the supernatant of culture broth and cell-free extracts of S. lividans, and both preparations showed the same characteristics. The N-terminal amino acid sequence of both KS1DHs was M-D-W-A-E-E-Y-D, which coincided with the amino acid sequence deduced from the nucleotide sequence. Thus, the extracellular enzyme may derived from leakage of S. lividans cells during cultivation rather than secretion by processing of the signal sequence. The molecular weight of the enzyme was about 55,000, identical with the size deduced from the nucleotide sequence (Mr 54,329). The optimum conditions for its activity were pH 10.0 and 40°C. The enzyme catalyzed the conversion of several 3-keto-steroids, but those containing 11α- or 11β-hydroxyl group were converted at low rates. The amino acid sequence of KS1DH from A. simplex is similar to those of KS1DH of Pseudomonas testosteroni and fumarate reductase from Shewanella putrefaciens.

Original languageEnglish
Pages (from-to)1043-1049
Number of pages7
JournalJournal of biochemistry
Volume117
Issue number5
DOIs
Publication statusPublished - 1995 May
Externally publishedYes

Keywords

  • 3-ketosteroid-δ1-dehydrogenase
  • Arthrobacter simplex
  • Streptomyces lividans

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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