title: Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4.
abstract: Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.
authors: Department of Biochemistry and Molecular Biology I, Universidad Complutense de Madrid, 28040 Madrid, Spain. laurfern@bio.ucm.es
authors: Laura
authors: L
authors: Fernández de las Heras
authors: Robert
authors: R
authors: van der Geize
authors: Oliver
authors: O
authors: Drzyzga
authors: Julián
authors: J
authors: Perera
authors: Juana
authors: J
authors: María Navarro Llorens
mesh_terms: D000730:Androstadienes / Q000378:metabolism; D000735:Androstenedione / Q000031:analogs & derivatives / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003470:Culture Media; D017353:Gene Deletion; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D000730:Androstadienes; D003470:Culture Media; D007527:Isoenzymes; C006573:1,4-androstadiene-3,17-dione; D000735:Androstenedione; D002784:Cholesterol; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase