The full-length cDNA (LeDET2) encoding a 257 amino acid protein homolog of Arabidopsis DET2 (AtDET2) was isolated in tomato (Lycopersicon esculentum). LeDET2 has 76% similarity with AtDET2 and structural characteristics conserved among plant and mammalian steroid 5-alpha-reductases (5Rs). LeDET2 is ubiquitously expressed in tomato tissues with higher levels in leaf than in stem, root, seed and callus. When expressed in mammalian cells (COS-7), recombinant LeDET2 was active on substrates typical of mammalian 5Rs (progesterone, testosterone, androstenedione), but reduced at very lowlevels campestenone, the substrate described for AtDET2. Similar results were obtained with the expression in COS-7 of recombinant AtDET2 that showed 5R activity for progesterone and not for campestenone. Recombinant LeDET2 was inhibited by several inhibitors of the human 5Rs and the application of an active inhibitor to tomato seedlings induced dwarfism and morphological changes similar to BR-deficient mutants. In tomato tissues, campestenone was 5-reduced in leaf, stem and root homogenates, like progesterone and testosterone, while androstenedione was converted to testosterone, evidencing for the first time a 17-hydroxysteroid dehydrogenase activity in plants. Moreover, two separate 5R activities with different kinetic characteristic and response to inhibitors were characterized in tomato tissues. The presence of two 5R isoenzymes was demonstrated also in Arabidopsis using the det2-1 mutant, in which a residual 5R activity for campestenone and progesterone was evidenced and characterized. Therefore, the existence of two isoenzymes of 5R is probably characteristic of the whole plant kingdom highlighting the similarities between the animal and plant steroid biosynthetic pathways.

5alpha-Reductase Activity in Lycopersicon Esculentum: Cloning and Functional Characterization of Ledet2 and Evidences of the Presence of Two Isoenzymes / FABIANA ROSATI; IRENE BARDAZZI; PAOLA DE BLASI; LISA SIMI; DINA SCARPI; ANTONIO GUARNA; MARIO SERIO; M. RACCHI; GIOVANNA DANZA. - In: JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY. - ISSN 0960-0760. - STAMPA. - 96:(2005), pp. 287-299. [10.1016/j.jsbmb.2005.04.035]

5alpha-Reductase Activity in Lycopersicon Esculentum: Cloning and Functional Characterization of Ledet2 and Evidences of the Presence of Two Isoenzymes

ROSATI, FABIANA;LISA SIMI;SCARPI, DINA;GUARNA, ANTONIO;SERIO, MARIO;RACCHI, MILVIA LUISA;DANZA, GIOVANNA
2005

Abstract

The full-length cDNA (LeDET2) encoding a 257 amino acid protein homolog of Arabidopsis DET2 (AtDET2) was isolated in tomato (Lycopersicon esculentum). LeDET2 has 76% similarity with AtDET2 and structural characteristics conserved among plant and mammalian steroid 5-alpha-reductases (5Rs). LeDET2 is ubiquitously expressed in tomato tissues with higher levels in leaf than in stem, root, seed and callus. When expressed in mammalian cells (COS-7), recombinant LeDET2 was active on substrates typical of mammalian 5Rs (progesterone, testosterone, androstenedione), but reduced at very lowlevels campestenone, the substrate described for AtDET2. Similar results were obtained with the expression in COS-7 of recombinant AtDET2 that showed 5R activity for progesterone and not for campestenone. Recombinant LeDET2 was inhibited by several inhibitors of the human 5Rs and the application of an active inhibitor to tomato seedlings induced dwarfism and morphological changes similar to BR-deficient mutants. In tomato tissues, campestenone was 5-reduced in leaf, stem and root homogenates, like progesterone and testosterone, while androstenedione was converted to testosterone, evidencing for the first time a 17-hydroxysteroid dehydrogenase activity in plants. Moreover, two separate 5R activities with different kinetic characteristic and response to inhibitors were characterized in tomato tissues. The presence of two 5R isoenzymes was demonstrated also in Arabidopsis using the det2-1 mutant, in which a residual 5R activity for campestenone and progesterone was evidenced and characterized. Therefore, the existence of two isoenzymes of 5R is probably characteristic of the whole plant kingdom highlighting the similarities between the animal and plant steroid biosynthetic pathways.
2005
96
287
299
FABIANA ROSATI; IRENE BARDAZZI; PAOLA DE BLASI; LISA SIMI; DINA SCARPI; ANTONIO GUARNA; MARIO SERIO; M. RACCHI; GIOVANNA DANZA
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/310308
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