Molecular cloning, expression in Streptomyces livdans, and analysis of a gene cluster from Arthrobacter simplex encoding 3‐ketosteroid‐Δ1‐dehydrogenase, 3‐ketosteroid‐Δ5‐isomerase and a hypothetical regulatory protein

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

Research output: Contribution to journalArticle

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 GylR, 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 GylR. 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
DOIs
Publication statusPublished - 1995 Mar
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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