Publications

• GhDET2, a steroid 5alpha-reductase, plays an important role in cotton fiber cell initiation and elongation.

  Luo M., Xiao Y., Li X., Lu X., Deng W., Li D., Hou L., Hu M., Li Y., Pei Y.

  Cotton (Gossypium hirsutum L.) fibers, one of the most important natural raw materials for textile industry, are highly elongated trichomes from epidermal cells of cotton ovules. DET2, an Arabidopsis steroid 5d-reductase, is considered to catalyze a major rate-limiting in brassinosteroid (BR) bios ... >> More

  Cotton (Gossypium hirsutum L.) fibers, one of the most important natural raw materials for textile industry, are highly elongated trichomes from epidermal cells of cotton ovules. DET2, an Arabidopsis steroid 5d-reductase, is considered to catalyze a major rate-limiting in brassinosteroid (BR) biosynthesis. To understand the role of BRs in cotton fiber development, GhDET2, which putatively encodes a steroid 5alpha-reductase by sequence comparison, was cloned from developing fiber cells. In vitro assessment of GhDET2 protein activity confirmed that GhDET2 encodes a functional steroid 5alpha-redutase. High levels of GhDET2 transcript were detected during the fiber initiation stage and the fiber rapid elongation stage. Antisense-mediated suppression of GhDET2 inhibited both fiber initiation and fiber elongation. Similarly, treating cultured ovules with finasteride, a steroid 5alpha-reductase inhibitor, reduced fiber elongation. Inhibition of fiber cell elongation by expression of antisense GhDET2 or the finasteride treatment could be reversed by epibrassinolide, a biologically active BR. Furthermore, seed coat-specific expression of GhDET2 increased fiber number and length. Therefore, GhDET2 and BRs play a crucial role in the initiation and elongation of cotton fiber cells, suggesting that modulation of BR biosynthesis factors may improve fiber quality or yield. << Less

  Plant J. 51:419-430(2007) [PubMed] [EuropePMC]

• Formation of dolichol from dehydrodolichol is catalyzed by NADPH-dependent reductase localized in microsomes of rat liver.

  Sagami H., Kurisaki A., Ogura K.

  The alpha-saturation reaction involved in the biosynthesis of dolichol has been investigated with rat liver preparations. Under improved in vitro conditions with 10,000 x g supernatant of rat liver homogenates in the presence of NADPH at pH 8.0, dolichol was synthesized from isopentenyl diphosphat ... >> More

  The alpha-saturation reaction involved in the biosynthesis of dolichol has been investigated with rat liver preparations. Under improved in vitro conditions with 10,000 x g supernatant of rat liver homogenates in the presence of NADPH at pH 8.0, dolichol was synthesized from isopentenyl diphosphate and Z,E,E-geranylgeranyl diphosphate. Neither dolichyl diphosphate nor dolichyl phosphate was detected. The chain length distribution of the dolicohol was the same as that of dehydrodolichyl products. In an assay system containing dehydrodolichol, dehydrodolichyl phosphate, or dehydrodolichyl diphosphate as a substrate, dehydrodolichol was predominantly converted into dolichol. The enzyme that catalyzes the conversion of dehydrodolichol to dolichol was localized in microsomes. The reductase activity was stimulated 9-fold by the addition of a 100,000 x g soluble fraction. The reductase had an opimal pH at 8.0. These results indicate that dolichol is formed from dehydrodolichol in rat liver microsomes. The formation of dolichol from dehydrodolichol was also catalyzed by 10,000 x g supernatant of rat or pig testis homogenates. << Less

  J. Biol. Chem. 268:10109-10113(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Arabidopsis det2 is defective in the conversion of (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one in brassinosteroid biosynthesis.

  Noguchi T., Fujioka S., Takatsuto S., Sakurai A., Yoshida S., Li J., Chory J.

  Previously, we have shown that the Arabidopsis det2 (deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid 5alpha-reductase) acts early in the proposed BR biosynthetic pathway. In this paper we present further biochemical characterization of det2. ... >> More

  Previously, we have shown that the Arabidopsis det2 (deetiolated2) mutant is defective in the biosynthesis of brassinosteroids (BR) and that DET2 (a steroid 5alpha-reductase) acts early in the proposed BR biosynthetic pathway. In this paper we present further biochemical characterization of det2. We have undertaken metabolic experiments with 2H-labeled substrates of intermediates involved in the formation of campestanol from campesterol, and quantitative analysis of intermediates in Arabidopsis wild type and det2. The results of these studies indicate the early operating steps of BR biosynthesis as: campesterol --> 4-en-3beta-ol --> 4-en-3-one --> 3-one --> campestanol in Arabidopsis, with det2 deficient in the conversion of 4-en-3-one to 3-one. We have also detected these intermediates in the formation of campestanol from campesterol and their metabolic conversions using cultured cells of Catharanthus roseus. These studies confirmed the biosynthetic sequence of events from campesterol to campestanol as was found in Arabidopsis. As such, the originally proposed biosynthetic pathway should be modified. << Less

  Plant Physiol. 120:833-840(1999) [PubMed] [EuropePMC]

• 5alpha-Reductase activity in Lycopersicon esculentum: cloning and functional characterization of LeDET2 and evidence of the presence of two isoenzymes.

  Rosati F., Bardazzi I., De Blasi P., Simi L., Scarpi D., Guarna A., Serio M., Racchi M.L., Danza G.

  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 5alpha-reductases (5alphaRs). LeDE ... >> More

  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 5alpha-reductases (5alphaRs). 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 5alphaRs (progesterone, testosterone, androstenedione), but reduced at very low levels campestenone, the substrate described for AtDET2. Similar results were obtained with the expression in COS-7 of recombinant AtDET2 that showed 5alphaR activity for progesterone and not for campestenone. Recombinant LeDET2 was inhibited by several inhibitors of the human 5alphaRs 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 5alpha-reduced in leaf, stem and root homogenates, like progesterone and testosterone, while androstenedione was converted to testosterone, evidencing for the first time a 17beta-hydroxysteroid dehydrogenase activity in plants. Moreover, two separate 5alphaR activities with different kinetic characteristic and response to inhibitors were characterized in tomato tissues. The presence of two 5alphaR isoenzymes was demonstrated also in Arabidopsis using the det2-1 mutant, in which a residual 5alphaR activity for campestenone and progesterone was evidenced and characterized. Therefore, the existence of two isoenzymes of 5alphaR is probably characteristic of the whole plant kingdom highlighting the similarities between the animal and plant steroid biosynthetic pathways. << Less

  J. Steroid Biochem. Mol. Biol. 96:287-299(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biochemical and pharmacogenetic dissection of human steroid 5 alpha-reductase type II.

  Makridakis N.M., di Salle E., Reichardt J.K.

  Human prostatic steroid 5alpha-reductase, encoded by the SRD5A2 gene on chromosome band 2p23, catalyses the irreversible conversion of testosterone to dihydrotestosterone (DHT), the most active androgen in the prostate, with NADPH as its cofactor. This enzyme has never been purified but a number o ... >> More

  Human prostatic steroid 5alpha-reductase, encoded by the SRD5A2 gene on chromosome band 2p23, catalyses the irreversible conversion of testosterone to dihydrotestosterone (DHT), the most active androgen in the prostate, with NADPH as its cofactor. This enzyme has never been purified but a number of competitive inhibitors have been developed for this enzyme since increased steroid 5alpha-reductase activity may cause benign prostatic hypertrophy and prostate cancer. We report here the detailed biochemical and pharmacogenetic dissection of the human enzyme by analysing 10 missense substitutions and three double mutants which are all naturally found in humans. Nine of these 13 mutants reduce activity (measured as Vmax) by 20% or more, three increase steroid 5alpha-reductase by more than 15% and one results in essentially unaltered kinetic properties suggesting that it is a truly neutral ('polymorphic') amino acid substitution. Substantial pharmacogenetic variation among the mutants was also observed when three competitive inhibitors, finasteride, GG745 (dutasteride) and PNU157706, were investigated. Our studies not only define the substrate and cofactor binding sites of human steroid 5alpha-reductase, but also have significant consequences for the pharmacological usage of steroid 5alpha-reductase inhibitors in human patients treated for prostatic conditions. << Less

  Pharmacogenetics 10:407-413(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Conservation of function between mammalian and plant steroid 5alpha-reductases.

  Li J., Biswas M.G., Chao A., Russell D.W., Chory J.

  Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares approximately 40% sequence identity with mammalian steroid 5alpha-reductases and is implica ... >> More

  Arabidopsis det2 mutants are small dark-green dwarfs displaying pleiotropic defects in light-regulated development during multiple stages of the plant life cycle. The DET2 gene encodes a protein that shares approximately 40% sequence identity with mammalian steroid 5alpha-reductases and is implicated in the synthesis of a class of plant steroids, the brassinosteroids. Here we show that the DET2 protein, when expressed in human embryonic kidney 293 cells, catalyzes the 5alpha-reduction of several animal steroid substrates and has similar kinetic properties to the mammalian steroid 5alpha-reductase enzymes. Moreover, human steroid 5alpha-reductases expressed in det2 mutant plants can substitute for DET2 in brassinosteroid biosynthesis. These data indicate that DET2 is an ortholog of the mammalian steroid 5alpha-reductases and provide further evidence that brassinosteroids play an essential role in light-regulated plant development. The structural and functional conservation between DET2 and human steroid 5alpha-reductases raise interesting issues concerning the evolutionary origin of the steroid hormone signaling system. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:3554-3559(1997) [PubMed] [EuropePMC]

• Tissue distribution and kinetic characteristics of rat steroid 5 alpha-reductase isozymes. Evidence for distinct physiological functions.

  Normington K., Russell D.W.

  The enzyme steroid 5 alpha-reductase (5 alpha-reductase) catalyzes the reduction of delta 4,5 double bonds in a variety of substrates and is thought to play both catabolic and anabolic roles in steroid hormone metabolism. Here, we describe the isolation and characterization of a cDNA encoding the ... >> More

  The enzyme steroid 5 alpha-reductase (5 alpha-reductase) catalyzes the reduction of delta 4,5 double bonds in a variety of substrates and is thought to play both catabolic and anabolic roles in steroid hormone metabolism. Here, we describe the isolation and characterization of a cDNA encoding the rat type 2 isozyme of 5 alpha-reductase and compare the kinetic properties and tissue-specific expression patterns of this isozyme with those of the type 1 isozyme. The type 2 isozyme has apparent Km values in the nanomolar range for steroid substrates, whereas the type 1 isozyme has micromolar affinities. The isozymes differ in their inhibition by various 4-azasteroids with the type 2 isozyme showing exquisite sensitivity (Ki = 40 pM) to 21,21-pentamethylene-4-aza-5 alpha-pregn-1-ene-3,20-dione. Messenger RNAs encoding the type 2 isozyme are more abundant than type 1 mRNAs in most male reproductive tissues, whereas the type 1 mRNAs predominate in peripheral tissues. Both 5 alpha-reductase mRNAs are more efficiently induced by dihydrotestosterone than by testosterone in the regenerating prostate. The differences in substrate affinities and tissue distributions of the 5 alpha-reductase isozymes suggest that type 2 plays an anabolic role and type 1 a catabolic role in the metabolism of androgens and other steroid hormones. << Less

  J. Biol. Chem. 267:19548-19554(1992) [PubMed] [EuropePMC]