MOLECULAR-CLONING, EXPRESSION IN STREPTOMYCES-LIVIDANS, AND ANALYSIS OF A GENE-CLUSTER FROM AUTHROBACTER SIMPLEX ENCODING 3-KETOSTEROID-DELTA(1)-DEHYDROGENASE, 3-KETOSTEROID-DELTA(5)-ISOMERASE AND A HYPOTHETICAL REGULATORY PROTEIN

The Arthrobacter simplex gene coding for 3-ketosteroid-Delta(1)-dehydr ogenase, a key enzyme in the degradation of the steroid nucleus, was c loned in Streptomyces lividans, Nucleotide sequence analysis revealed that the gene for 3-ketosteroid-Delta(1)-dehydrogenase (ksdD) is clust ered with at least two more genes possibly involved in steroid metabol ism, Upstream of ksdD, we found a gene, ksdR, encoding a hypothetical regulatory protein that shows homologies to KdgR, the negative regulat or of pectin biodegradation in Erwinia, and GylR, the activator for gl ycerol metabolism in Steptomyces. A helix-turn-helix DNA-binding domai n can be predicted at similar positions near the N-terminal of KsdR, K dgR and GylR, ksdl adjoining downstream to ksdD codes for a protein th at has strong similarities to 3-ketosteroid-Delta(5)-isomerases, The h ighly 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-Delta(1)-dehydrogenase of Pseudomonas tes tosteroni 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. livid ans strains carrying the ksdD gene overexpressed 3-ketosteroid-Delta(1 )-dehydrogenase. The expression of 3-ketosteroid-Delta(5)-isomerase, h owever, was barely detectable in recombinant S. lividans strains carry ing the ksdl gene, or in the parental Arthrobacter strain,