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

Summary

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.