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 (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

39 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)391-399
Number of pages11
JournalMolecular Microbiology
Volume15
Issue number5
DOIs
Publication statusPublished - Mar 1995
MoE publication typeA1 Journal article-refereed

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