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Originally published In Press as doi:10.1074/jbc.M604431200 on July 11, 2006

J. Biol. Chem., Vol. 281, Issue 37, 27264-27277, September 15, 2006
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Molecular and Enzymatic Characterizations of Novel Bifunctional 3beta-Hydroxysteroid Dehydrogenases/C-4 Decarboxylases from Arabidopsis thaliana*Formula

Alain Rahier{ddagger}1, Sylvain Darnet{ddagger}, Florence Bouvier{ddagger}, Bilal Camara{ddagger}, and Martin Bard§2

From the {ddagger}Institut de Biologie Moléculaire des Plantes, CNRS UPR2357, 28 Rue Goethe, 67083 Strasbourg Cedex, France and the §Biology Department, Indiana-Purdue University, Indianapolis, Indiana 46202

Received for publication, May 9, 2006 , and in revised form, June 30, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases (3betaHSD/D) involved in sterol synthesis, termed At3betaHSD/D1 and At3betaHSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3betaHSD/D activity, with the At3betaHSD/D1 isoform or with At3betaHSD/D2 isoform containing a C-terminal At3betaHSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3betaHSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3betaHSD/D isoenzymes showed similar substrate specificities that required free 3beta-hydroxyl and C-4-carboxyl groups but were quite tolerant in terms of variations of the sterol nucleus and side chain structures. Data obtained with 4{alpha}-carboxy-cholest-7-en-3beta-ol and its 3{alpha}-deuterated analog revealed that 3{alpha}-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3betaHSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3beta-hydroxy-4beta,14-dimethyl-5{alpha}-ergosta-9beta,19-cyclo-24(241)-en-4{alpha}-carboxylic acid, consistent with a decrease in 3betaHSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sterols are essential components of all eukaryotic cell membranes, and the biosynthetic pathways differ significantly. The sterol molecule becomes functional only after removal of the two methyl groups at C-4. Both methyl groups at C-4 are removed early and successively in animals and yeast, whereas in higher plants one methyl group is initially removed from a 4,4-dimethyl-9beta,19-cyclopropylsterol precursor, and the second is eliminated several steps later (1-3). In plants, we have characterized the activities of a sterol C-4 methyl oxidase (SMO),3 a 4{alpha}-carboxysterol-3beta-hydroxysteroid dehydrogenase/C-4 decarboxylase (3betaHSD/D), and an NADPH-dependent 3-oxosteroid reductase from partially purified preparations (4-6) in order to define the steps involved in C-4 demethylation in plants. The first step is initiated by the SMO, whereby this enzyme converts the C-4{alpha} methyl group to produce a 4{alpha}-carboxysterol derivative that is subsequently oxidatively decarboxylated by the 3betaHSD/D to produce a C-4-monodemethylated 3-oxosteroid, which is then stereospecifically reduced by the 3-ketoreductase. In contrast to animals and yeast where the SMO is encoded by a single gene (7), we biochemically characterized two distinct microsomal SMOs in Zea mays (4), and we identified two distinct families of SMO genes in Arabidopsis thaliana (8). Until now, the gene(s) coding the single bifunctional protein 3betaHSD/D (Fig. 1) have not been characterized.

Furthermore, little is known about plant hydroxysteroid dehydrogenases that are either members of the short chain dehydrogenases/reductases (SDR) (9-11) or the aldo-keto reductase family (12), which include soluble and membranebound HSDs. Identification and characterization of gene(s)-encoding plant HSD of the SDR family have not been reported thus far.

To further our knowledge about hydroxysteroid dehydrogenases and the sterol C-4 demethylation multienzymatic complex in plants, in particular, we have identified and characterized at the molecular and enzymatic levels two novel 3betaHSD/D isoforms from A. thaliana. In this study, we present identification and cloning of these 3betaHSD/Ds cDNAs, in vivo heterologous and functional expression in yeast, in vitro enzymological characterization of the recombinant isoenzymes, and in planta down-regulation by virus-induced gene silencing.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
All materials were purchased from Sigma or as otherwise specified in the text.

Strains and Plasmids—The erg26 SDG200 strain of Saccharomyces cerevisiae, deficient in 3beta-hydroxydehydrogenase/C-4 decarboxylase activity (Mata ade5, his3, leu2-3, ura3-52, erg26{Delta}::TRP1 trp1::hisG {Delta}hem1) used in the present study, has been described previously (13). Sterol auxotrophs were grown aerobically at 30 °C on solid enriched medium (YPG: 1% yeast extract, 2% peptone, 2% glucose) supplemented with 2% ergosterol or cholesterol dissolved in ethanol/Tween 80 (1:1, v/v) or on minimal medium (YNB: 0.67% yeast nitrogen base, 2% glucose) containing suitable supplements (50 mg/liter each), casamino acids (1 g/liter) and 2% of ergosterol or cholesterol. In the case of liquid medium, the concentration of sterol used was 0.5%. Sterol prototrophic strains were grown aerobically at 30 °C on solid or liquid YNB medium containing suitable supplements (50 mg/liter each) or enriched medium (YPG) in the presence of {delta}-aminolevulinic acid (50 mg/liter).


Figure 1
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  		FIGURE 1.
Reaction catalyzed by 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases and putative mechanism.

 
The pVT102U (14) S. cerevisiae shuttle vector optimized for expressing recombinant proteins in yeast was used for cloning, sequencing, and transformation of the erg26 strain. This plasmid contains an Escherichia coli origin of replication, a yeast 2-µm origin of replication, an E. coli ampicillin resistance gene, and the yeast URA3 gene. It contains an expression cassette, including the alcohol dehydrogenase promotor and terminator. TTO vector is an RNA viral vector and has been described previously (15). It consists of sequences from tobacco mosaic virus strain U1 (TMV-U1) and tomato mosaic virus (fruit necrosis strain F; ToMV-F).

Plants—Nicotiana benthamiana was grown in a greenhouse at 24 °C with a 16-h light/8-h dark cycle. A. thaliana plantlets were from the Wassilewskija ecotype.

cDNAs Cloning—We searched the Arabidopsis genome for genes encoding putative orthologs of the yeast ERG26 gene and also belonging to the hydroxysteroid dehydrogenase (HSD) family. Two genes were selected as follows: At1g47290 and At2g26260, and their corresponding cDNAs (At3betaHSD/D1 and At3betaHSD/D2, respectively) were cloned by PCR using reverse-transcribed mRNAs from the Wassilewskija ecotype. The amplified cDNA fragments were cloned into the XbaI and XhoI sites of the pVT102U shuttle vector and placed under the control of the constitutive alcohol dehydrogenase promoter. Because the lack of a C-terminal ER retrieval signal KKXX in At3betaHSD/D2 might result in inefficient localization in the ER yeast C-4 demethylation complex, we replaced the seven terminal amino acids of At3betaHSD/D2 with the KKID sequence to produce {Delta}-At3betaHSD/D2, which was also cloned into the pVT102U vector.

Total RNAs from A. thaliana plantlets were extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's protocol and treated with DNase to remove any residual DNA. SuperScript II kit (Invitrogen) was used to perform RT-PCRs with the following reverse primers: 5'-TTAGCTGATCTTCTTGCTCCCGAACACTTTC-3' (P2) corresponding to At1g-47290 (NM_179448 [GenBank] ) and 5'-CTAATTGAAATATATGGTCATACCCTTTCGGCCC-3' (P4) corresponding to At2g26260 (NM_128183 [GenBank] .1), to specifically amplify the corresponding cDNAs. PCR amplification using an aliquot of RT-PCR, with the primer pair P1 = 5'-ATGGAAGTTACAGAGACTGAGCGATGGTGC-3' and P2, yielded a 1143-bp cDNA, namely At3betaHSD/D1 (AY957470 [GenBank] ) with two nucleotides changes between At3betaHSD/D1 (ecotype Wassilewskija) and the At1g-47290 gene transcript (ecotype Columbia) (NM_179448 [GenBank] ), leading to two altered amino acids at position 68 (Pro to Ser) and position 354 (Arg to Lys) probably reflecting the distinct ecotypes. Amplification with primers P3 = 5'-ATGTCGCCGGCAGCTACGGAAC-3' and P4 yielded a 1173-bp cDNA, namely At3betaHSD/D2 (DQ302749 [GenBank] ), identical to the initially annotated At2g26260 gene sequence (NM_128183 [GenBank] .1). At-tempts to amplify a cDNA corresponding to the recently updated splicing of the At2g26260 gene sequence (NM_128183 [GenBank] .2) with specific oligonucleotides were unsuccessful. A 1161-bp DNA termed {Delta}-At3betaHSD/D2, encoding an At3betaHSD/D2 derivative that was missing the last 7 amino acid residues from the C terminus which was replaced by three KID residues and tailed by the KKID sequence, was generated by PCR. Two primers were designed as follows. The forward primer was P5 = 5'-ATAATATCTAGAATGTCGCCGGCAGCTAC-3' containing an XbaI site, and the reverse primer was P6 = 5'-ATAATACTCGAGTTAGTCGATCTTCTTTCCGCCTCCAAGAATCC-3' containing an XhoI site. The At3betaHSD/D2 cDNA served as the template. PCR amplification entailed 30 cycles of 45 s at 94 °C, 30 s at 55 °C, and 90 s at 72 °C using the HiFi PCR Master (Roche Applied Science). The amplified fragments were cloned into the XbaI and XhoI sites of the pVT102U vector to generate the plasmids pVT-At3betaHSD/D1, and pVT-{Delta}-At3betaHSD/D2. Both strands of the amplified cDNAs were sequenced to ensure sequence fidelity.

To amplify 3betaHSD/D in N. benthamiana, primers were designed according to consensus regions of aligned putative 3betaHSD/D sequences of Solanacae and the codon usage of Nicotiana. A 500-bp fragment of the N. benthamiana 3beta-hydroxysteroid-dehydrogenase-C-4 decarboxylase geneamino acid residues from the C terminus was (Nb3betaHSD/D) (AM236597 [GenBank] ) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (DXR) (AM236596 [GenBank] ), which was used as a control, were PCR-amplified from RNAs extracted from leaves of N. benthamiana using the following forward primers, respectively: 5'-GCCTCGAGGCTGATTTGGGTCCATCCATTAAACTTGAG-3',5'-GCCTCGAGGGAGTGGCTATCAAAAGAAAGGAG-3', and the following reverse primers: 5'-TCCCTAGGTCCTGCCTTTGCAGCTGCAACTAATGAAGGAAC-3', 5'-TCCCTAGGCTTTATTGGCCAAGGCAATGTCCTTTC-3'. These products were cloned into the XhoI-AvrII restricted TTO viral vector (16) to generate TTO-Nb3betaHSD/D and TTO-NbDXR, respectively.

Virus-induced Gene Silencing in N. benthamiana—TTO-Nb3betaHSD/D and TTO-NbDXR constructs were used to inoculate young N. benthamiana plants as described previously (16). To measure silencing of Nb3betaHSD/D, semi-quantitative reverse transcription was performed also as described previously (8, 17). Total RNAs isolated from uninoculated TTO-NbDXR and TTO-Nb3betaHSD/D leaves were extracted and used for PCR. The forward 5'-GGAAGAGGCTTTGCTGCTCGG-3' and reverse 5'-GCATGTGCTACATTCTCCACG-3' primers that anneal outside the region used for Nb3betaHSD/D silencing were employed. Amplification of the N. benthamiana {alpha}-tubulin gene was performed as a control using the forward 5'-ATGCTTTCATCATATGCCCCTGTG-3' and reverse 5'-CAGCACCAACTTCCTCGTAATC-3' primers.

Identity Scores and Phylogenetic Analysis—Protein sequences of Arabidopsis 3betaHSD/D and their homologs were identified by BLAST searches and aligned using the ClustalW algorithm. A neighbor-joining tree was constructed based on the sequence alignment and tested further with 1000 bootstraps resampling by using the MacVector package.

Transformations—S. cerevisiae transformations were performed using the lithium acetate procedure as described previously (18). The transformed erg26 yeast strain was plated on minimal YNB medium containing suitable supplements (adenine, 50 µg ml-1) without uracil and 2% of ergosterol, as well as on the same minimal YNB medium without uracil but also containing {delta}-aminolevulinic acid (50 mg/liter). Cells were grown aerobically at 30 °C.

Sterol Analysis—Lyophilized yeast cells (10-30 mg) were sonicated in the presence of KOH/methanol (6%, w/v) (2 ml) for 10 min and heated in the same medium at 70 °C under reflux conditions for 2 h. The mixture was diluted with 1 volume of water, and after acidification to pH 3, total sterols were extracted three times with 3 volumes of ethyl acetate. The extract was dried on Na2SO4, evaporated to dryness, treated with 500 µl of a 0.4 M solution of diazomethane in diethyl ether for 1 h at 0°C and 1 h at room temperature, and then evaporated to dryness. Sterols were analyzed by gas chromatography. GC analysis was carried out with a Varian GC model 8300 (Les Ulis, France) equipped with a flame ionization detector at 300 °C, column injector at 250 °C, and a fused capillary column (WCOT, 30 m x 0.25 mm inner diameter) coated with DB1(H2 flow rate of 2 ml/min). The temperature program used included a 30 °C/min increase from 60 to 240 °C and followed by a 2 °C/min increase from 240 to 280 °C. Relative retention times (tR) are given with respect to cholesterol (tR = 1). Identification of individual sterols was performed using a GC-MS spectrometer (Agilent 5973N) equipped with an "on column" injector and a capillary column (30 m x 0.25 mm inner diameter) coated with DB5. Sterols were unequivocally identified by retention times and an electron impact spectrum identical to that of authentic standards (19).

Lyophilized plantlets of N. benthamiana (0.9-2.0 g) were homogenized with an Ultra-Turrax homogenizer in the presence of methanol/methylene chloride (2:1, v/v) and heated in the same medium at 70 °C under reflux conditions for 3 h. The mixture was filtered, and the extract was evaporated to dryness. Acidic derivatives (RF = 0.0-0.12), desmethylsterols (RF = 0.27), 4{alpha}-methylsterols (RF = 0.38), 4,4-dimethylsterols (RF = 0.44), and sterol esters (RF = 0.70) were purified twice by TLC on Silica Gel 60F254 plates (Merck), using methylene chloride as developing solvent, and various fractions were eluted from the silica gel. Sterol esters were saponified under standard conditions. The polar fraction (RF = 0-0.12) was treated with diazomethane at 0 °C and subsequently acetylated under standard conditions. The derivatized polar fraction was further purified by TLC on Silica Gel 60F254 plates (Merck), using methylene chloride as developing solvent. The fraction with an RF = 0.07-0.20 and migrating with the standard of 4-carbomethoxysteryl acetate was eluted from the silica gel. A known amount of cholesterol was added as internal standard for GC quantification in the case of the 4{alpha}-methylsterol, 4,4-dimethylsterol, and 4-carboxymethylsteryl acetate fractions and of coprostane in the case of the desmethylsterol fraction. All fractions were then analyzed by GC and GC-MS under the same conditions as described above.

Chemical Details—Melting points are uncorrected. Proton magnetic resonance was monitored in a [2H]chloroform solution with a Brucker 400- or 500-MHz spectrometer. Chemical shifts ({delta}) (ppm) were determined relative to tetramethylsilane. Coupling constants (J) were in Hertz.

Substrates—For 4{alpha}-carboxy-cholest-7-en-3beta-ol (1) (Fig. 2) was synthesized as described previously (5). The melting point was 231-233 °C; MS m/z (relative intensity) M+ = 430(36), 412(10), 397(5), 386(100), 317(2), 299(19), 273(19), 271(19), 255(63), 229(22), 213(18). For 1H NMR: {delta} 0.533 (3H, s, H18), 0.844 (3H, s, H19), 0.862 (3H, d, J = 6.6, H26 or H27), 0.867 (3H, d, J = 6.6, H26 or H27), 0.918 (3H, d, J = 6.5, H21), 2.030 (3H, dt, J = 11, J = 4, H6beta), 2.364 (1H, dd, J = 11, H4beta), 3.809 (1H, m, H3{alpha}), 5.131 (1H, S, {omega}1/2 = 10, H7).

For synthesis of 3{alpha}-deutero-4{alpha}-carboxy-cholest-7-en-3beta-ol (11), a solution of 4{alpha}-carbomethoxy-cholest-7-en-3-one (120 mg) (synthesized as described previously (5) in absolute methanol (20 ml)) was stirred with sodium deuteroborohydride (NaBD4) (Aldrich) (25 mg) for 2 h at 0 °C. After the usual work up, the solid residue was purified by TLC (SiO2, eluted with hexane/ethyl acetate, 75:25 v/v) to yield pure 3{alpha}-deutero-4{alpha}-carbomethoxy-cholest-7-en-3beta-ol (RF = 0.18) (55 mg), which was then separated from its 3{alpha}-hydroxy epimer (RF = 0.44) and crystallized in methanol, m.p. 147-149 °C. The compound showed a single peak in GC (tR = 1.368, DB5). MS m/z (relative intensity) was as follows: M+ = 445(95), 427(100), 412(54), 385(84), 367(11), 352(83), 314(77), 313(37), 272(38), 254(29). The deuteration was 0% d0, 100% d1. For 1H NMR, {delta}: 0.528(3H, s, H18), 0.832(3H, s, H19), 0.862(3H, d, J = 6.6, H26 or H27), 0.867(3H, d, J = 6.6, H26 or H27), 0.916(3H, d, J = 6.5, H21), 2.337(1H, d, J = 11.4, H4beta), 3.717 (3H, s, OMe), 5.110(1H, d, J = 3.6, H7). This compound (30 mg) was refluxed with a solution of KOH (100 mg) in ethanol/H2O (80:20, v/v) (5 ml) for 2 h at 75 °C. After cooling at room temperature, the mixture was poured into water pH 3 (40 ml) and extracted with ethyl acetate. The solvent was dried over Na2SO4 and evaporated to dryness to give a white solid powder that was recrystallized from ethyl acetate to yield pure 3{alpha}-deutero-4{alpha}-carboxy-cholest-7-en-3beta-ol (11) (25 mg) m.p. 229-231 °C. The trimethylsilyl derivative of (11) showed a single peak in GC (DB1). For MS of the trimethylsilyl derivative: m/z (relative intensity) M+ = 575(5), 560(7), 485(6), 470(5), 457(33), 368(16), 367(19), 352 (100), 325(45), 254(16). The deuteration was 0% d0, 100% d1. 1H NMR of (11): {delta}: 0.533 (3H, s, H18), 0.842 (3H, s, H19), 0.863 (3H, d, J = 6.6, H26 or H27), 0.868 (3H, d, J = 6.6, H26 or H27), 0.918 (3H, d, J = 6.5, H21), 2.032 (3H, dt, J = 12.6, J = 2.4, H6beta), 2.355 (1H, dd, J = 11.5, H4beta), 5.134 (1H, d, J = 2.4, H7).


Figure 2
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  		FIGURE 2.
Chemical structures of the compounds considered in the present work.

 
Synthesis of 4{alpha}-carbomethoxy-cholest-7-en-3beta-ol (10) and 4{alpha}-carboxy-cholest-7-en-3{alpha}-ol (5). Reduction of 4{alpha}-carbomethoxy-cholest-7-en-3-one by sodium borohydride (NaBH4) and TLC purification under the same conditions as described above yielded 4{alpha}-carbomethoxy-cholest-7-en-3beta-ol (10)(RF = 0.18) and 4{alpha}-carbomethoxy-cholest-7-en-3{alpha}-ol (21) (RF = 0.44). (10) was crystallized from methanol as follows: m.p 152-154 °C. It showed a single peak in GC (tR = 1.368, DB5). MS m/z (relative intensity) M+ = 444(41), 426(64), 411(46), 384(69), 367(12), 351(100), 313(66), 271(31), 253(22). For 1H NMR: {delta}: 0.529(3H, s, H18), 0.833(3H, s, H19), 0.863(3H, d, J = 6.6, H26 or H27), 0.868(3H, d, J = 6.6, H26 or H27), 0.917(3H, d, J = 6.5, H21), 2.341(1H, dd, J = 10.3, J = 10.3, H4b), 3.719(3H, s, COOCH3), 3.802(1H, m, H3{alpha}), 5.111(1H, d, J = 3.7, H7). Compound (21) showed a single peak in GC (tR = 1.333, DB5). MS m/z (relative intensity) M+ = 444(100), 426(37), 411(31), 384(27), 367(14), 351(76), 313(56), 271(19), 253(22), 211(29). Saponification of (21) with KOH in ethanol/H2O as above yielded pure 4{alpha}-carboxy-cholest-7-en-3{alpha}-ol (5). 1H NMR of (5): {delta}: 0.534(3H, s, H18), 0.838(3H, s, H19), 0.865(3H, d, J = 6.6, H26 or H27), 0.871(3H, d, J = 6.6, H26 or H27), 0.922 (3H, d, J = 6.5, H21), 2.507(1H, dd, J = 12.0, J = 2.2, H4beta), 4.194(1H, s, {omega}1/2 = 8 Hz, H3beta), 5.150(1H, d, J = 3.3, H7). The 4{alpha}-carbomethoxy derivative of (5) showed a single peak in GC (tR = 1.334, DB5); MS: m/z (relative intensity) M+ = 444(100), 426(45), 411(40), 384(23), 367(10), 351(79), 313(55), 271(25), 253(22), 21 (18).

For 4{alpha}-carboxy-4beta-methyl-cholest-8,24-dien-3beta-ol (2), this carboxysterol was extracted and purified from the erg26 yeast strain growing on YPG-enriched liquid medium supplemented with 0.5% of ergosterol and in the presence of {delta}-aminolevulinic acid (50 mg/liter) following the standard procedures described above. Crystallization in ethyl acetate yielded pure (2) (6.3 mg) m.p. 227-229 °C. MS of the 4{alpha}-carbomethoxy derivative of (2): m/z (relative intensity) M+ = 456(100), 441(39), 438(17), 423(17), 396(17), 363(22), 285(22), 225(15). 1H NMR of (2): {delta}: 0.588(3H, s, H18), 0.939(3H, d, J = 6.4, H21), 1.008(3H, s, H19), 1.176(3H, s, H4beta), 1.600(3H, s, H26), 1.680(3H, s, H27), 4.021(1H, dd, J = 16, J = 7.5, H3{alpha}), 5.090 (1H, tt, J = 7.1, J = 1.3, H24).

For [241-3H]4{alpha}-carboxy-4beta,14{alpha}-dimethyl-ergosta-9beta,19-cyclo-24(241)-en-3beta-ol (3)(1 µCi, 2.5 Ci/mol) and [242-3H]4{alpha}-carboxy-5{alpha}-stigmasta-7,24(241)-dien-3beta-ol (4) (2 µCi, 2.5 Ci/mol), these compounds were prepared and purified as described in Ref. 4 by enzymatic 4{alpha}-methyl oxidation of [241-3H]4-methylenecycloartanol (13) and [242-3H]24-ethylidenelophenol (14), respectively, using a maize microsomal extract in the presence of NADPH. They showed a single radioactive band on SiO2-TLC using ethyl acetate as developing solvent.

[241-3H]4,4,14{alpha}-trimethyl-9beta,19-cyclo-ergost-24(241)-en-3beta-ol (24-methylenecycloartanol) (13), [242-3H]4{alpha}-methylstigmasta-7,24(241)-dien-3beta-ol (24-ethylidenelophenol) (14) were enzymatically synthesized and purified as described in Ref. 4. They showed a single peak in GC and a single radioactive band on SiO2-TLC using methylene chloride as developing solvent. They were diluted with cold material to the desired specific radioactivity (2-5 Ci/mol). [14C]4,4-Dimethyl-cholest-8,24-dien-3beta-ol (12) was isolated from the mutant erg25-25c grown in the presence of [2-14C]acetate as described in Ref. 20 and showed more than 98% GC and TLC purity. Specific radioactivity was 1.5 Ci/mol.

Pregnenolone (16), trans-androstenone (17), 3beta-androstanediol (18), and dehydroepiandrosterone (19) were purchased from Sigma.

Preparation of Microsomes—Yeast microsomes were prepared as described previously (20). The corresponding 100,000 x g supernatants corresponding to the cytosolic extracts were concentrated 5-8-fold by dialysis over carboxymethylcellulose sodium salt (Fluka) for 16 h at 4 °C.

Standard Assay for Recombinant 4{alpha}-Carboxysterol-3-dehydrogenase-C-4 Decarboxylases —Microsomes (0.8 mg of protein) were incubated in the presence of exogenous synthetic 4{alpha}-carboxy-5{alpha}-cholest-7-en-3beta-ol (1) (20-200 µM) emulsified with Tween 80 (final concentration 1.5 g/liter) and NAD+ at a 400 µM concentration (which is 50-fold the Km value). Incubations were continued aerobically at 30 °C with gentle stirring for 30-45 min. During this period the progression of the reaction was linear. The reaction was stopped by adding 0.5 ml of EtOH. After addition of a known amount of coprostanone (1-4 µg) as internal standard, sterols and sterones were extracted from the incubation mixture three times with a total volume of 15 ml of n-hexane, and after drying with Na2SO4, the extract was concentrated to dryness. Under these conditions, the residual carboxysterol substrate was not extracted. The extract was analyzed by TLC on silica gel eluted with CH2Cl2 (developed twice). The fraction migrating as authentic standards of coprostanone and cholest-7-en-3-one (6) and containing the enzymatically produced cholest-7-en-3-one (RF = 0.50) was eluted and analyzed by GC (DB1) (supplemental Fig. A). The amount of cholest-7-en-3-one (6) produced (tR = 1.074) was calculated by comparison of the integrated peak areas with a known amount of added coprostanone (tR = 1.000), which also allowed the reaction rate of cholest-7-en-3-one to be determined. No endogenous component having the same tR as (6) was present in the inactivated control. The ketone metabolite produced by the reaction was unequivocally identified by its retention time on GC and by an electron impact mass spectrum identical to that of an authentic synthetic standard (Table 1 and supplemental Fig. A). Moreover, in control experiments, GC-MS analysis of the complete extract before TLC analysis indicated the absence of formation of cholest-7-en-3beta-ol during the course of the incubation, confirming that in the absence of exogenously added NADPH to the microsomal extract, the C-4-demethylated-3-oxo-derivative was not reduced and further metabolized in accord with the cofactor requirements for this reduction step (6). Under these conditions, the estimated limit of detection of the 3betaHSD/D activity was 0.1-0.2 nmol x h-1·mg-1. Incubation of the 3{alpha}-deuterated substrate (11) was performed under the same standard conditions. For control experiments, the untransformed substrate (11) was extracted from the incubation mixture with ethyl acetate and methylated with diazomethane. GC-MS analysis of the 4-carbomethoxy derivative revealed no lost of deuterium, thus excluding a possible washout of the deuterium label at C-3 during the incubation procedure.


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  		TABLE 1
GC-MS analysis of enzymatic products by the recombinant 3betaHSD/Ds from Arabidopsis

 
Apparent maximum velocity V and V/K values were determined by fitting the data to the Michaelis-Menten equation using the nonlinear regression program DNRP-EASY derived by Duggleby which corresponds to Ref. 21. Computer-assisted linear regression analysis gave correlation coefficients greater than 0.98 (n = 5-6). The primary deuterium kinetic isotope effects were defined as DV = HVmax/DVmax and D(V/K) = H(Vmax/Km)/D(Vmax/Km) according to the conventions of Northrop (22, 23).

Incubation of 4{alpha}-Carboxysterol Analogs and Identification of Enzyme-generated Products—The apparent Km and Vmax of analogs (1, 5, and 2) (Tables 3 and 4) were determined by incubating them for 30-45 min at 30 °C under standard assay conditions in a microsomal preparation containing either 3betaHSD/D1 or {Delta}3betaHSD/D2. The concentration of substrate was 20-150 µM, and the concentration of NAD+ was 0.4 mM (50-fold the measured Km value). As indicated previously, in the absence of NADPH addition to the microsomal extract, the expected C-4-demethylated 3-oxo-derivative metabolites were not further metabolized. In the case of substrates (3 and 4) for which apparent Km and Vmax values could not be determined because of difficulties in obtaining sufficient quantities of these two substrates, the reaction rates of (3 and 4) were compared with those of (1, 2, and 5) in microsomal extracts containing either 3betaHSD/D1 or {Delta}3betaHSD/D2. Low substrate concentrations (40 µM) were used to determine enzyme specificity; the concentration of NAD+ was 0.4 mM, and the reaction rates measured for (2, 3, 4, and 5) were normalized to the rate obtained with the standard substrate (1) (taken as 100%, Fig. 4, A and B). In the case of the carboxysterols (2, 3, 4, and 5) after addition of coprostanone as internal standard, extraction, and TLC analysis, the fraction migrating between a standard of 4-demethyl-sterone (RF = 0.50) and 4,4-dimethyl-sterone (RF = 0.70) was eluted and analyzed by GC and GC-MS (Table 1). The 3betaHSD/D products were unequivocally identified by their retention time and an electron impact spectrum identical to that of authentic standards (Table 1). The data were compared with those obtained in the case of a control where the microsomes were inactivated. In addition, in the case of incubation of radiolabeled carboxysterols (2, 3, and 4), the newly labeled sterone was directly visualized by radioscanning of the TLC plate, in accord with the migration of the corresponding nonlabeled standards. The areas of the GC peak of coprostanone and of the product peak formed, corrected from endogenous components of the same tR (if present) determined in the corresponding control, allowed the rate of transformation of these analogs to be measured. Incubations of labeled sterols (12, 13, and 14) and corresponding control incubation with inactivated enzyme were extracted according to the same procedure. Radioscanning of the TLC analysis indicated the complete recovery of the added substrate and absence of metabolic alteration of these compounds by any of the two recombinant At3betaHSD/Ds microsomal preparations. In the case of the carboxymethylated analog (10), the hexane extracts of the incubation and control samples were directly analyzed by GC and gave identical profiles, indicating the absence of detectable At3betaHSD/D activity with this analog.


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  		TABLE 3
Apparent kinetics parameters for selected substrates of recombinant Arabidopsis 3betaHSD/D1 For each experiment, kinetic parameters of the different substrates were measured in the same microsomal preparation.

 


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  		TABLE 4
Apparent kinetics parameters for selected substrates of recombinant Arabidopsis {Delta}3betaHSD/D2 For each experiment, kinetic parameters of the different substrates were measured in the same microsomal preparation.

 
Similarly, the extracts of incubations of 3beta-hydroxysteroids (16, 17, 18, and 19) and ursolic acid (15) and corresponding controls were directly analyzed by GC and GC-MS. Comparison to the corresponding controls performed with inactivated microsomes revealed the absence of dehydrogenated product and complete recovery of these compounds, which was confirmed by ion monitoring that corresponded to the masses of the substrate and the expected C-3 dehydrogenated product.

Miscellaneous—Membrane protein was determined as described by Bradford (24).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of 3betaHSD/D in Arabidopsis—We searched the Arabidopsis genome for genes encoding putative orthologs of the yeast ERG26 gene and belonging to the HSD family. Among the best candidates were At1g47290 and At2g26260, two genes that shared the highest sequence identity with the yeast 3betaHSD/D protein Erg26p (30 and 29% respectively). Corresponding cDNAs (named At3betaHSD/D1 for A. thaliana 3beta-hydroxy-steroid dehydrogenase/decarboxylase isoform 1 and At3betaHSD/D2, respectively) were cloned by PCR using reverse-transcribed mRNAs from the Wassilewskija ecotype. Sequencing of At3betaHSD/D1 (AY957470 [GenBank] ) indicated an open reading frame of 1143 bp with two nucleotide changes from the At1g47290 gene transcript (ecotype Columbia, NM_179448 [GenBank] ) leading to two amino acid changes. Sequencing of At3betaHSD/D2 (DQ302749 [GenBank] ) indicated an open reading frame of 1173 bp that was identical with the annotated At2g26260 gene transcript (NM_128183 [GenBank] .1). While this study was in progress, an additional possible splicing site of the At2g26260 gene leading to a 3'-extended sequence (1695 bp) was annotated (NM_128183 [GenBank] .2); however, we were not able to amplify the corresponding cDNA from our RNAs preparation.

Sequences Analysis—At3betaHSD/D1 (381 amino acids) and At3betaHSD/D2 (391 amino acids) proteins share 80% identity (supplemental Fig. B). A sequence comparison analysis revealed significant homology between the two At3betaHSD/Ds to the 3beta-hydroxysteroid dehydrogenase/isomerase protein family (Pfam 01073), a member of the SDR superfamily (FAD/NAD(P)-binding Rossmann fold superfamily clan) (9-11, 25, 26). However, there is little sequence identity (only 15-30%) between different SDR enzymes. The two sequences include N-terminal conserved glycine and aspartic residues, TGGXGXXAX18D, which are required to form the coenzyme-binding site. In addition, the At3betaHSD/D1 and At3betaHSD/D2 proteins possess the YX3K motif (159YX3K163 for At3betaHSD/D1) conserved in the active site of most of the members of the SDR family (10) (supplemental Fig. B). Finally a conserved serine residue, found at position 130 or 131 for At3betaHSD/D1, might constitute the third residue of the catalytic triad proposed for a variety of members of the SDR family (9, 10, 27). In the At3betaHSD/D1 and At3betaHSD/D2 proteins, there is a single putative membrane spanning domain near the C terminus (supplemental Fig. B). Finally, At3betaHSD/D1 possesses a C-terminal ER retrieval signal KKXX, which is absent in At3betaHSD/D2.

Phylogenetic Analysis of the At3betaHSD/D—A molecular phylogenetic tree of the amino acid sequences of a variety of characterized HSD proteins from different organisms related to the putative At3betaHSD/Ds was designed (Fig. 3). The protein sequences of At3betaHSD/D1 and At3betaHSD/D2 show 30% identity with the yeast ERG26 protein (13) and 37% identity with the NAD(P)H steroid dehydrogenase-like protein from animals (28). These clones show 22% identity with the human 3beta-hydroxysteroid dehydrogenase/isomerase (29-30) and 26% identity with the cholesterol dehydrogenase from Nocardia (31), all enzymes catalyzing dehydrogenation of 3beta-hydroxysteroids. The similarity of the two At3betaHSD/Ds to Erg26p and Nsdhl is higher than with any other known 3betaHSD. Together with the present 3betaHSD/Ds from plant, these orthologs from yeasts and animals form a differentiated cluster distinct from other HSDs, particularly those catalyzing dehydrogenations at positions other than the 3beta of the steroid nucleus or displaying an opposite stereochemistry for C-3 hydride abstraction. These data are consistent with the suggestion that At3betaHSD/D1 and At3betaHSD/D2 code two plant 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases isoenzymes.


Figure 3
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  		FIGURE 3.
Phylogenetic analysis of 3betaHSD/Ds. A rooted neighbor-joining tree was constructed by using the 11beta-hydroxysteroid dehydrogenase from Mus musculus as an out group. Bootstrap values are indicated adjacent to the branches. Candida albicans ERG26 (AF329471); S. cerevisiae ERG26 (P53199); Homo sapiens Nsdhl (Q15738); M. musculus Nsdhl (AF100198); A. thaliana 3betaHSD/D1 (AY957470); A. thaliana 3betaHSD/D2 (DQ302749); N. benthamiana 3betaHSD/D (AM236597); Nocardia cholesterol oxidase (D90244); Mycobacterium tuberculosis probable cholesterol dehydrogenase (CAA17222); H. sapiens 3beta-hydroxysteroid dehydrogenase/isomerase (NM000198); Rattus norvegicus 20{alpha}-hydroxysteroid dehydrogenase (P51652); R. norvegicus 3{alpha}-hydroxysteroid dehydrogenase (P23457); H. sapiens 3{alpha}-hydroxysteroid dehydrogenase type II (NP_003730); H. sapiens 20{alpha}-hydroxysteroid dehydrogenase (NP_001344); H. sapiens 3{alpha}-hydroxysteroid dehydrogenase type III (NP_001345); Drosophila melanogaster 7{alpha}-hydroxysteroid dehydrogenase (NP_523396); M. musculus 17beta-hydroxysteroid dehydrogenase (NP_034605); M. musculus 11beta-hydroxysteroid dehydrogenase NP_032314.

 
At3betaHSD/D1, and {Delta}-At3betaHSD/D2 Containing the At3betaHSD/D1 ER Retrieval Signal, Can Complement a Yeast Strain Deficient in Erg26p—To further characterize the function of At3betaHSD/D1 and At3betaHSD/D2, we performed a yeast complementation assay in the ERG26-deficient strain SDG200 deficient in 3betaHSD/D activity (13). In S. cerevisiae, the ERG26 product is an essential enzyme because of the fact that disruption of ERG26 is lethal, and the erg26 strain requires ergosterol or cholesterol supplementation for viability (13). Despite one putative membrane-embedded domain, the lack of the C-terminal ER retrieval signal KKXX in At3betaHSD/D2 might result in inefficient localization in the ER yeast C-4 demethylation complex. Thus, we replaced the seven terminal amino acids of At3betaHSD/D2 with the KKID sequence to produce {Delta}-At3betaHSD/D2 (387 amino acids). The At3betaHSD/D1 and {Delta}-At3betaHSD/D2 cDNAs were cloned into the pVT102U shuttle vector under the control of the constitutive alcohol dehydrogenase promoter.

The pVT transformants were plated on complementation selection plates in a medium supplemented with {delta}-aminolevulinic acid required for heme synthesis but devoid of sterol. Heme auxotrophy was required to facilitate sterol uptake for erg26 mutants. The erg26 strain transformed with pVT-At3betaHSD/D1 and pVT-{Delta}-At3betaHSD/D2 was capable of growing aerobically without ergosterol supplementation, whereas erg26/pVT-VOID transformants could grow only on an ergosterol-supplemented medium (supplemental Fig. C). To confirm the authenticity of the complementation by At3betaHSD/D1 and {Delta}-At3betaHSD/D2, several colonies of the prototrophic strains were picked from the selection plate and grown in an {delta}-aminolevulinic acid-containing liquid medium devoid of sterol. After sterol extraction, the sterol profiles were analyzed by GC and GC-MS. The strains erg26-pVT-At3betaHSD/D1 and erg26-pVT-{Delta}-At3betaHSD/D2 accumulated more than 70% of C-4-demethylated sterols, including ergosterol (50-52%) as the major sterol, and minor amounts (12-16%) of residual 4,4-dimethylsterols (Table 2). In comparison the auxotrophic strain erg26/pVT-VOID grown in a cholesterol-containing media accumulated lanosterol and small amounts of 4{alpha}-carboxy-4,14-dimethyl-cholest-8-en-3beta-ol (20) as shown previously (13). In the presence of {delta}-aminolevulinic acid, erg26/pVT-VOID accumulated 4{alpha}-carboxy-4beta-methyl-cholest-8-en-3beta-ol (2) but no ergosterol as shown previously for erg26 (13). These results demonstrate that the A. thaliana At3betaHSD/D1 and {Delta}-At3betaHSD/D2 cDNAs can efficiently complement the ergosterol auxotroph yeast erg26 strain by restoring growth and endogenous ergosterol synthesis.


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  		TABLE 2
Sterol composition of mutant erg26 transformed with different plasmids Values represent the means ± S.D. of the analysis of three to five clones.

 
At3betaHSD/D1 and {Delta}-At3betaHSD/D2 Have 3beta-Hydroxysteroid Dehydrogenase/C-4 Decarboxylases Activity in Vitro—We next performed an enzymatic assay to test whether the two recombinant Arabidopsis putative 3betaHSD/D proteins in the transformed erg26 strain indeed possess 3-hydroxysteroid dehydrogenase/C-4 decarboxylase activity. Overexpression and purification of plant membrane proteins for functional analysis are still relatively unexplored fields for yeast or bacteria with little documentation in the literature. Additionally, in the case of an enzyme that is part of a membrane-bound multienzymatic complex, interactions with other components of the complex may be necessary for optimum enzymatic activity. Thus, 3betaHSD/D activity was assayed in the microsomal extracts and corresponding cytosolic fractions prepared from erg26-pVT-VOID, erg26-pVT-At3betaHSD/D1, and erg26-pVT-{Delta}-At3betaHSD/D2 by using the standard assay conditions for recombinant 3betaHSD/D described under "Experimental Procedures." The results from these studies revealed that microsomal extracts obtained from erg26-pVT-At3betaHSD/D1 and erg26-pVT-{Delta}-At3betaHSD/D2 were able to oxidatively decarboxylate the 3betaHSD/D substrate, 4{alpha}-carboxycholest-7-en-3beta-ol (1), with a high efficiency in the presence of NAD+, to produce a single 3-keto-4-decarboxylated metabolite, cholest-7-en-3-one (6), which was unequivocally identified by GC-MS analysis (Table 1 and supplemental Fig. A). 3betaHSD/D activity was undetectable in reactions with microsomal extracts of erg26/pVT-VOID. All corresponding concentrated cytosolic extracts were also inactive. The observed high catalytic competence of At3betaHSD/D1 and {Delta}-At3betaHSD/D2 revealed that they indeed encode two membrane-bound plant 3betaHSD/Ds.

Determination of the Apparent Kinetic Parameters of At3betaHSD/D1 and {Delta}-At3betaHSD/D2 for 4{alpha}-Carboxy-cholest-7-en-3beta-ol (1) and Its 3{alpha}-Deuterated Analog (11)—In two separate series of experiments, the apparent kinetic parameters of each 3betaHSD/D with substrate 4{alpha}-carboxy-cholest-7-en-3beta-ol (1) were determined by varying the concentration of (1) by using a constant 50-fold Km concentration of NAD+. However, the Vmax for NAD+ could not be measured due to the inability of using saturating concentrations of carboxysterol (1), because it has limited solubility in aqueous media. Under our standard assay conditions, the velocity/substrate concentration curves obey simple Michaelis-Menten kinetics with respect to (1) and NAD+ cofactor (supplemental Fig. C). The obtained kinetics data for 3betaHSD/D1 and -2 are summarized in Tables 3 and 4, respectively.

To gain insight into the enzyme mechanism of the two 3betaHSD/Ds, we compared Vmax and V/K values measured for 3{alpha}-protonated 4{alpha}-carboxy-cholest-7-en-3beta-ol (1) with those obtained with its synthetic 3{alpha}-deuterated analog (11) in the same microsomal preparation. We used a noncompetitive assay, in which the protonated and deuterated substrates are measured separately. The advantage of the direct comparison method is that it is the only means to determine the primary kinetic deuterium isotope effect on Vmax (DV). For both At3betaHSD/D1 and {Delta}-At3betaHSD/D2, the measured primary deuterium kinetic isotopic effect for (11) was near unity, both for V and V/K, within experimental error (Tables 3 and 4 and supplemental Fig. D). Moreover, recovery and GC-MS analysis of the untransformed substrate (11) revealed no lost of deuterium, thus excluding any washout of the deuterium label at C-3 during the incubation procedure.


Figure 4
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  		FIGURE 4.
Substrate preference of recombinant Arabidopsis 3betaHSD/D1 (A) and {Delta}3betaHSD/D2 (B). The specificity of each 3betaHSD/D was analyzed separately in the corresponding microsomal preparation. For each isoenzyme, the reaction rates of the various substrates at a 40 µM concentration were measured in the same enzymatic preparation and compared with the rate obtained with 4{alpha}-carboxy-cholest-7-en-3beta-ol (1) which was taken as 100%. 100% corresponds to a rate of 20.5 nmol·mg-1·h-1 (A) and 100% corresponds to a rate of 22.5 nmol·mg-1·h-1 (B). Values represent the mean of two measurements with an S.D.<10% of single substrate activity measurement. 4{alpha}-Carboxy-4beta-methyl-3beta-hydroxysterols are as follows: 4{alpha}-carboxy-4beta-methyl-cholest-8,24-dien-3beta-ol (2); 4{alpha}-carboxy-4beta,14{alpha}-dimethyl-9beta,19-cyclo-ergost-24(241)-en-3beta-ol (3). 4{alpha}-Carboxy-3beta-hydroxysterol is as follows: 4{alpha}-carboxy-stigmasta-7,24(241)-dien-3beta-ol (4). 4{alpha}-Carboxy-3{alpha}-hydroxysterol is as follows: 4{alpha}-carboxy-cholest-7-en-3{alpha}-ol (5). 4{alpha}-Substituted-3beta-hydroxysterols are as follows: 4{alpha}-carbomethoxy-cholest-7-en-3beta-ol (10); 4,4-dimethyl-cholest-8,24-dien-3beta-ol (12); 4,4,14{alpha}-trimethyl-9beta,19-cyclo-ergost-24(241)-en-3beta-ol (13); 4{alpha}-methyl-stigmasta-7,24(241)-dien-3beta-ol (14). 3beta-Hydroxysteroids are as follows: ursolic acid (15); pregnenolone (16); trans-androstenone (17); 3beta-androstanediol (18); dehydroepiandrosterone (19).

 
Substrate Screening of the At3betaHSD/Ds—We used a series of natural or synthetic substrate analogs to determine the structural requirements of the 3betaHSD/Ds. The steroid substrate screen used our standard 3betaHSD/D assay conditions (see "Experimental Procedures") at saturating levels of NAD+ cofactor (50-fold the Km value). The assay conditions were complemented by analysis of products and residual substrates using GC-MS.

First, a series of 3beta-hydroxy-4{alpha}-carboxysterols with distinct nucleus or side chain structures were assayed with 3betaHSD/D1. These included (3) and (4), the respective products of the two plant sterol C-4-methyl oxidases (SMO) (4), and thus the most probable substrates for decarboxylation in plants, and (2) the physiological substrate of the yeast 3betaHSD/D, which accumulates in the erg26 mutant (13, 32), was also assayed. Because of the difficulty of obtaining sufficient quantities of (3) and (4) that could only be produced enzymatically in vitro, we could not determine the kinetic constants of these two analogs but were able to compare their reaction rates, in the same 3betaHSD/D1 enzymatic preparation, with that of the other substrates used at the same concentration. The data indicate that all four 3beta-hydroxy-4-carboxysterols (1-4) were dehydrogenated and decarboxylated by At3betaHSD/D1 (Fig. 4A and Table 3) and that this isoenzyme did not show a marked preference for any of these four 3beta-hydroxy-4-carboxysterols. The corresponding enzymatic 3-oxo-C-4-decarboxylated products of reaction were unequivocally identified as (6-9), respectively, by GC-MS analysis (Table 1).

To probe the stereoselectivity of the plant 3betaHSD/D1 for the 3beta-hydroxy group of the substrate, the epimer of (1) with the 3{alpha}-configuration (5) was synthesized. It was dehydrogenated by the recombinant 3betaHSD/D1, but with a 50-fold lower activity, to also produce (6), which was unequivocally identified by GC-MS (Table 3 and Fig. 4A).

Next, we examined the requirement for the C-4-carboxylic substituent. Ursolic acid (15), a triterpene derivative possessing a 3beta-hydroxyl group and a distal C-17-carboxyl function, was not metabolized. Converting the carboxyl group in (1) to generate the C-4-carboxymethylated derivative (10), or substitution of the carboxyl group by a methyl group as in the 4,4-dimethylsterols (12) and (13) or the 4{alpha}-methylsterol (14), totally abolished the dehydrogenase activity of 3betaHSD/D1 in the presence of NAD+ cofactor (Fig. 4A). Indeed, following incubation of cold or radioactive labeled samples of these analogs, TLC and GC-MS analysis of the reaction excluded the formation of the corresponding 3-oxo products. Moreover, by using a series of 3beta-hydroxysteroids, pregnenolone (16), trans-androstenone (17), 3beta-androstanediol (18), and dehydroepiandrosterone (19), which are known substrates of a number of animal 3betaHSDs, we could not detect any oxidative conversion of the 3beta-OH by the plant 3betaHSD/D1.

Similarly, the structural requirements of {Delta}3betaHSD/D2 were examined in a separate series of experiments by comparing the reaction rates of the substrate analogs in the {Delta}3betaHSD/D2 enzymatic preparation. The data (Fig. 4B and Table 4) indicated that all four 3beta-hydroxy-4-carboxysterols (1-4) were dehydrogenated and decarboxylated by {Delta}3betaHSD/D2. {Delta}3betaHSD/D2 did not show a marked preference for any of these substrates other than a substantial lower transformation rate observed for (2), the physiological substrate of the yeast 3betaHSD/D (which is however not synthesized in plants). The {Delta}3betaHSD/D2 isoenzyme also dehydrogenated (5) the 3{alpha}-epimer of (1) at a 50-fold lower rate and was also not able to metabolize compounds (10 and 12-19).


Figure 5
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  		FIGURE 5.
Silencing phenotypes and semi-quantitative RT-PCR analysis of Nb3betaHSD/D N. benthamiana plants. Photographs of uninfected N. benthamiana (A), N. benthamiana plants infected with TTO-NbDXR (B), and TTO-Nb3betaHSD/D (C) were taken 3 weeks postinoculation. D, RT-PCR products of Nb3betaHSD/D and {alpha}-tubulin were analyzed using gel electrophoresis with ethidium bromide staining. {alpha}-Tubulin was amplified as a control transcript. The experiment was performed three times with identical results.

 
Silencing of Endogenous N. benthamiana 3betaHSD/D—In order to learn more about the function of the 3betaHSD/D, a cDNA fragment of 500 bp termed Nb3betaHSD/D, homologous to the N terminus of At3betaHSD/D1 and At3betaHSD/D2 (or {Delta}-At3betaHSD/D2), was isolated in N. benthamiana and cloned into the viral TTO vector. This sequence was placed under the control of the tobacco mosaic virus (TMV-U1) coat protein subgenomic promoter in the antisense orientation. Nb3betaHSD/D shares 59-57% identity with At3betaHSD/D1 and At3betaHSD/D2 and thus falls clearly within the 3betaHSD/D family (Fig. 3 and supplemental Fig. B).

N. benthamiana plants were inoculated with infectious TTO-Nb3betaHSD/D mRNAs (Fig. 5C), and noninfected plants (Fig. 5A) as well as plants infected with the previously described TTO-DXR (Fig. 5B) construct (17) were used as controls. Silencing of Nb3betaHSD/D strongly reduced the growth of young leaves (Fig. 5C) compared with uninfected plants and plants infected with TTO-NbDXR (Fig. 5, A and B). To measure the VIGS of 3betaHSD/D, semiquantitive RT-PCR analysis was performed using 3betaHSD/D primers annealing outside of the region used for the silencing. We observed a 70% reduction of the transcripts in Nb3betaHSD/D silenced plants compared with uninfected plants and the TTO-NbDXR control (Fig. 5D).

Approximately 3 weeks after infection, total lipids from young plants, containing sterols and expected carboxysterol derivatives, were extracted and purified by TLC allowing the separation of steryl esters, 4,4-dimethyl-, 4{alpha}-methyl-, 4-demethylsterols, and a more polar fraction which was treated with diazomethane to stabilize and decrease the polarity of the carboxysterol derivatives. The sterols were quantified by GC and compared with authentic standards by GC-MS electron impact mass spectra.

Results indicate that the most noteworthy difference between the control and the TTO-Nb3betaHSD/D infected plants appeared to be the addition of a novel sterol derivative that represents 2-3% of the total sterol content and is not detectable in extracts from control plants (Fig. 6). This compound was identified as 4{alpha}-carboxy-4beta,14{alpha}-dimethyl-9beta, 19-cyclo-ergost-24(241)-en-3beta-ol (3) by GC-MS analysis after derivatization and comparison with the MS spectrum of an authentic enzymatically produced sample (4). In addition, a 2-fold accumulation of 4,4-dimethylsterols was observed in the plants infected with TTO-Nb3betaHSD/D, whereas the bulk of the sterol profile remained unchanged within the limit of experimental error (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The SDR family includes more than 2000 annotated enzymes that contain a 250-350-amino acid residue core structure, catalyzing NAD(P)(H)-dependent oxidoreductions of great functional diversity (9-11, 33). Despite a sequence identity level of only 15-30% between different SDR members, the three-dimensional folds are quite similar, except for the C-terminal regions. According to the Sanger Institute data base, most of the 3beta-hydroxysteroid dehydrogenases show a common 3betaHSD architecture (Pfam 01073) found in the present At3betaHSD/D isoforms, and a small number (5 of 198) possess an additional C-terminal 200-amino acid residue segment homologous to the reticulon containing two large hydrophobic regions that are likely embedded in the ER membrane (34, 35). Whereas the 3betaHSD/D1 cDNA corresponding to the At2g47290 gene encodes a protein that shows the 3betaHSD architecture, the 3betaHSD/D2 cDNA encodes a protein corresponding to the 3betaHSD core domain of the At2g26260 gene product possessing the 3betaHSD-reticulon architecture.


Figure 6
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  		FIGURE 6.
GC-MS analysis of diazomethane-treated and TLC-purified polar lipid extracts from N. benthamiana infected with TTO1-Nb3betaHSD/D reveals the presence of a novel derivative, 4{alpha}-carboxy-4beta,14{alpha}-dimethyl-9beta,19-cyclo-ergost-24(241)-en-3beta-ol (A). GC-MS analysis indicated that none of the other peaks present in the control or the infected plants corresponded to a C-4-carboxysterol derivative. The control experiment corresponds to plants infected with TTO-NbDXR. The experiment was performed twice with two independent set of plants and gave identical results.

 
The two Arabidopsis clones, as well as the Nicotiana 3betaHSD/D, share the typical signatures of the SDR family (supplemental Fig. B), including a nonclassical N-terminal glycine-rich motif TGGXGXXA, in which the third Gly residue is replaced by Ala. This nonclassical motif is generally found in the SDR group belonging to multifunctional enzyme complexes (11). In addition, the highly conserved YX3K segment (residues 159-163 in 3betaHSD/D1), assigned to the catalytic center (36), is found in both At3betaHSD/D1, -D2, and Nb3betaHSD/D. Therefore, based on the sequence alignment of the plant 3betaHSD/Ds with members of the SDR family (Pfam 01073), the conserved residues Ser-131, Tyr-159, and Lys-163 found in At3betaHSD/D1, -D2, and Nb3betaHSD/D could constitute the consensus catalytic triad, Ser, Tyr, Lys, proposed for SDR catalysis, facilitating the initial oxidation of (1) and analogs and requiring NAD+ as coenzyme (9-10, 36).

The 3betaHSD/D proteins show low sequence identity with other members of the SDR family thus forming a differentiated cluster in the phylogenetic tree (Fig. 3). The formation of this distinct subfamily could reflect the remarkable bifunctionality of the 3betaHSD/D proteins.

Identification of genes encoding membrane proteins can be achieved using the technique of "functional complementation" in a relevant yeast deletion mutant (37, 38). Complementation of the yeast erg26 mutant by plant 3betaHSD/D cDNAs inserted into a vector optimized for yeast expression was expected to restore both growth in the absence of ergosterol and endogenous ergosterol biosynthesis. At3betaHSD/D1 and {Delta}-At3betaHSD/D2 restored growth, high levels of ergosterol biosynthesis, and in vitro high catalytically competent 3betaHSD/Ds localized exclusively in the corresponding microsomal extracts. In contrast to most SDR proteins, including HSDs, biochemical studies performed in animals (39), yeast (40), and plants (5) have shown that native 3betaHSD/D is membrane-bound. Although a single hydrophobic portion was found in the Arabidopsis 3betaHSD/D1 and {Delta}-At3betaHSD/D2 proteins, the activities of both recombinant At3betaHSD/Ds containing an ER retrieval signal were found in the corresponding microsomal extracts. These data indicate that At3betaHSD/D1 and {Delta}-At3betaHSD/D2 are functionally inserted in the yeast C-4-demethylation enzymatic complex and are able to channel endogenous 4{alpha}-carboxy-4beta-methylcholest-8,24-dien-3beta-ol (2) through this complex and the sterol pathway.

Extensive studies performed in yeast have elucidated the protein-protein interactions with regard to sterol biosynthesis and particularly the C-4 demethylation complex (40, 41). Functional association of At3betaHSD/Ds with the C-4 demethylation complex could involve a limited number of amino acid interactions, including charged pairing between the partners as shown in a number of membrane-bound electron transfer complexes such as the cytochrome b5-cytochrome b5 reductase (42, 43) or the cytochrome P450-cytochrome P450 reductase (44) complexes. Mutagenesis of the At3betaHSD/Ds should be studied to probe which amino acids are necessary for these interactions.

Examination of the apparent kinetic parameters (Tables 3 and 4) of (1) for the At3betaHSD/Ds revealed Km values that are in the same order of magnitude to those of a variety of native post-squalene sterol biosynthetic enzymes from yeast (20) and native (2) or recombinant (45) plant sterol enzymes. In contrast, the apparent rates of At3betaHSD/D1 and {Delta}-At3betaHSD/D2 (66-86 nmol·mg-1·h-1), as well as that of the wild type diploid yeast Sc3betaHSD/D (366 nmol·mg-1·h-1; data not shown), appear relatively high in comparison to the rates of other enzymes of the post-squalene sterol pathway measured in the corresponding microsomal preparations (2), including other components of the C-4 demethylation complex such as the SMO from yeast (Vm = 1.2 nmol·mg-1·h-1) (20) or plants (Vm = 8.1 nmol·mg-1·h-1) (4). These data suggest that 3betaHSD/D is not rate-limiting for the overall process of C-4 demethylation and that 4-carboxysterols are probably transient intermediates. This is consistent with the observation that they have not been isolated in plant or wild type yeast thus far.

The high catalytic activity of {Delta}-3betaHSD/D2 (Table 4) indicates that the 200-amino acid C-terminal hydrophobic segment (homologous to reticulon present in the complete At2g26260 gene product and absent in {Delta}-3betaHSD/D2) is not involved in 3betaHSD/D catalysis. In addition, the calculated molecular mass of 3betaHSD/D1 and {Delta}-3betaHSD/D2, 42 kDa for both, is in agreement with the apparent molecular mass of 45 kDa found for the native and catalytically competent 3betaHSD/D purified from microsomal extracts of Z. mays (5).

Deuterium kinetic isotope effects have been used extensively to study the mechanism of numerous enzymes and particularly those involving a hydrogen transfer step (for review see Refs. 22, 23, and 46-48). We recently used kinetic isotope effects to study the mechanism of a yeast recombinant plant membrane sterol desaturase (49); however, no such study has been carried out with an HSD thus far. In noncompetitive assays, the primary deuterium kinetic isotope effects for oxidation of 3{alpha}-deutero-4{alpha}-carboxy-cholest-7-en-3beta-ol (11) catalyzed by 3betaHSD/D1 and {Delta}-3betaHSD/D2 were near unity, i.e. DV = 1.10; D V/K = 0.93-1.03. Similarly, we did not observe any significant kinetic isotope effect with a microsomal preparation of wild type yeast Sc3betaHSD/D (data not shown). This lack of an isotope effect indicates that the 3{alpha}-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. One possibility to explain the lack of an isotope effect would be that one or several steps of the reaction pathway, including chemical and product release, might be slower than the hydride transfer step and thus completely masked the DV. Another possibility is that (11) behaves as a sticky substrate that reacts to give products as fast or faster than it dissociates from the enzyme, i.e. the enzyme-substrate complex has a high commitment to catalysis (47, 48, 50).

Extensive studies using multiple isotope effects have been carried out with a number of enzymes catalyzing oxidative decarboxylations with NADP+ as cosubstrate, exemplified by the malic enzyme (51-53) or the 6-phosphogluconate dehydrogenase (54). In these cases, a stepwise mechanism was shown, and a small isotope effect for the hydride transfer step was measured, comparable with that found with the 3betaHSD/Ds studied here. However, measurement of a single isotope effect is not sufficient to elucidate the mechanism of the 3betaHSD/Ds, although it would seem logical that 3C-H bond cleavage and decarboxylation are separate events. Determination of the mechanism of the 3betaHSD/Ds will require further studies, including in particular the 13C isotope effect for C-C bond cleavage (53, 54), and variation with pH of primary isotope effects (47, 55-57).

To complete our catalytic analysis, we studied the substrate requirements of each of the two 3betaHSD/Ds in a separate series of experiments incubating various substrate analogs with enzymatic preparations. For both 3betaHSD/Ds isoenzymes, the data showed a substrate specificity that required free 3beta-hydroxyl and C-4 carboxyl groups indicating that both recombinant At3betaHSD/Ds are highly specific for 3beta-hydroxy-4{alpha}-carboxysterols. In contrast, substrate requirements were quite tolerant in terms of variations of the sterol nucleus and side chain structures. Remarkably, the data did not reveal for either of the two 3betaHSD/Ds a clear preference between substrates possessing one or two substituents at C-4. In addition, the substrate preferences of the two 3betaHSD/Ds were found to be very similar. This is in contrast with the strict and distinct structural requirements observed for the two distinct SMOs metabolizing 4,4-dimethyl- and 4{alpha}-methylsterols, respectively, to produce the corresponding carboxysterols that are the physiological substrates of the present 3betaHSD/Ds (4). Furthermore, (2), the typical substrate of the yeast 3betaHSD/D, was efficiently oxidized by both At3betaHSD/Ds, whereas 4,4-dimethyl-zymosterol (12), the substrate of the yeast SMO, was not metabolized by any of the plant SMOs (20). These data suggest that a single type of 3betaHSD/D is metabolizing the products of the two distinct SMOs inside the plant C-4 demethylation complex.

The VIGS experiments carried out in this study led to young N. benthamiana plants exhibiting a clear biochemical phenotype. As this biochemical phenotype was observed in several distinct infected plants but not in plants infected with the TTO-DXR construct or noninfected plants, it can clearly be ascribed to the presence of the Nb3betaHSD/D cDNA. These biochemical changes obtained after infection with TTO-Nb3betaHSD/D, correlating with the specific reductions in Nb3betaHSD/D mRNA levels, confirm the genetic silencing of the corresponding Nb3betaHSD/D endogene.

Silencing of Nb3betaHSD/D resulted in a substantial accumulation of a novel 4{alpha}-carboxysterol, 4{alpha}-carboxy-4beta,14{alpha}-dimethyl-9beta,19-cyclo-ergost-24(241)-en-3beta-ol (3), involved in the pathway for removing the first methyl group at C-4, thereby confirming in planta the function of the present 3betaHSD/Ds. Recent studies in A. thaliana indicate that plants compromised early in the sterol biosynthetic pathway, i.e. upstream of the removal of the second C-4 methyl group, such as the fackel mutant defective in C-14 reductase (58, 59), show severe defects in development and embryogenesis, whereas those affected later in the pathway, such as dwf7/ste1 (60) or dwf1/DIM (61), do not. Thus, in the event of an impaired expression of a gene upstream of the second C-4 demethylation step, plants might develop regulatory processes to reduce these defects, thereby limiting as in the present case the accumulation of (3). In these lines, a limited accumulation of the substrates of the SMOs in SMO-silenced plants was also observed previously by using the same VIGS approach in N. benthamiana (8). In this respect, 3betaHSD/D-silenced plants, albeit accumulating modest amounts of (3), present substantial developmental alterations that will need further work to be precisely characterized.

The present combination of molecular and biological chemical approaches allowed a thorough identification and functional characterization of 3betaHSD/D as one further step in the completion of the molecular inventory of sterols synthesis in higher plants. Genetic redundancy or lethality may account for the failure to isolate plant 3betaHSD/D genes by screening of phenotypes by using a genetic approach. Given that plant mutants affected in 3betaHSD/D have not been reported thus far, the present study provides important clues for the physiological roles of C-4-substituted sterols in photosynthetic eukaryotes. Finally, these two novel oxidative decarboxylases constitute the first plant hydroxysteroid dehydrogenase genes of the SDR superfamily to be molecularly and enzymatically characterized.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY957470 [GenBank] , DQ302749 [GenBank] , AM236597 [GenBank] , and AM236597 [GenBank] .

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. A-D. Back

2 Supported by National Institutes of Health Grant GM62104. Back

1 To whom correspondence should be addressed. Tel.: 33390241861; Fax: 33390242002; E-mail: enzymo{at}bota-ulp.u-strasbg.fr.

3 The abbreviations used are: SMO, sterol C-4-methyl oxidase; 3betaHSD/D, 3beta-hydroxysteroid dehydrogenase/C-4 decarboxylases; ER, endoplasmic reticulum; SDR, short chain dehydrogenases/reductases; HSD, hydroxysteroid dehydrogenase; GC-MS, gas chromatography-mass spectrometry; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We are indebted to Annie Hoeft for GC-MS analysis and helpful technical assistance. We thank Monto Kumagai and Large Scale Biology Corp. for the TT0 vector. We are grateful to J. D. Sauer for recording the NMR spectra.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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