Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter simplex encoding 3-ketosteroid-AΔ1-dehydrogenase, 3-ketosteroid-Δ5-isomerase and a hypothetical regulatory protein

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

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38 Citations (Scopus)

Abstract

The Arthrobacter simplex gene coding for 3-ketosteroid-Δ1-dehydrogenase, a key enzyme in the degradation of the steroid nucleus, was cloned in Streptomyces lividans. Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-Δ1-dehydrogenase (ksdD) is clustered with at least two more genes possibly involved in steroid metabolism. Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulator of pectin biodegradation in Erwinia, and GyIR, the activator for glycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domain can be predicted at similar positions near the N-terminal of KsdR, KdgR and GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-Δ5-isomerases. The highly conserved Tyr and Asp residues are present in the active-centre motif of the enzyme. The translated ksdD gene product was found to be similar to the 3-ketosteroid-Δ1-dehydrogenase of Pseudomonas testosteroni and to the fumarate reductase of Shewanella putrefaciens. A region highly conserved between the two steroid dehydrogenases can be aligned to the active-centre motif of the fumarate reductase. S. lividans strains carrying the ksdD gene overexpressed 3-ketosteroid-Δ1-dehydrogenase. The expression of 3-ketosteroid-Δ5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.

Original languageEnglish
Pages (from-to)895-905
Number of pages11
JournalMolecular Microbiology
Volume15
Issue number5
Publication statusPublished - 1995 Mar
Externally publishedYes

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ASJC Scopus subject areas

  • Molecular Biology
  • Microbiology

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