Molecular cloning, expression in Streptomyces lividans, and analysis of a gene cluster from Arthrobacter 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-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-delta 1-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 GyIR, 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 GyIR. ksdl adjoining downstream to ksdD codes for a protein that has strong similarities to 3-ketosteroid-delta 5-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-delta 1-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-delta 1-dehydrogenase. The expression of 3-ketosteroid-delta 5-isomerase, however, was barely detectable in recombinant S. lividans strains carrying the ksdl gene, or in the parental Arthrobacter strain.
Molnár
I
I
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Japan.
Choi
K P
KP
Yamashita
M
M
Murooka
Y
Y
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D007664:Ketosteroids / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017421:Sequence Analysis; D013251:Steroid Isomerases / Q000737:chemistry / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics*
D004268:DNA-Binding Proteins; D007664:Ketosteroids; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase; D013251:Steroid Isomerases; C022660:steroid delta-isomerase