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

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.