Molecular and Enzymatic Characterizations of Novel Bifunctional 3β-Hydroxysteroid Dehydrogenases/C-4 Decarboxylases from Arabidopsis thaliana

Abstract

We have isolated two cDNAs from Arabidopsis thaliana encoding bifunctional 3␤-hydroxysteroid dehydrogenase/C-4 decarboxylases (3␤HSD/D) involved in sterol synthesis, termed At3␤HSD/D1 and At3␤HSD/D2. Transformation of the yeast ergosterol auxotroph erg26 mutant, which lacks 3␤HSD/D activity, with the At3␤HSD/D1 isoform or with At3␤HSD/D2 isoform containing a C-terminal At3␤HSD/D1 endoplasmic reticulum-retrieval sequence restored growth and ergosterol synthesis in erg26. An in vitro enzymatic assay revealed high 3␤HSD/D activity for both isoenzymes in the corresponding microsomal extracts. The two At3␤HSD/D isoenzymes showed similar substrate specificities that required free 3␤-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␣-carboxy-cholest-7-en-3␤-ol and its 3␣-deuterated analog revealed that 3␣-hydrogen-carbon bond cleavage is not the rate-limiting step of the reaction. In planta reduction on the expression of the 3␤HSD/D gene as a consequence of VIGS-mediated gene silencing in Nicotiana benthamiana led to a substantial accumulation of 3␤-hydroxy-4␤,14-dimethyl-5␣-ergosta-9␤,19-cyclo-24(24 1 )-en-4␣-carboxylic acid, consistent with a decrease in 3␤HSD/D activity. These two novel oxidative decarboxylases constitute the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants.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-9␤,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␣-carboxysterol-3␤-hydroxysteroid dehydrogenase/C-4 decarboxylase (3␤HSD/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␣ methyl group to produce a 4␣-carboxysterol derivative that is subsequently oxidatively decarboxylated by the 3␤HSD/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 3␤HSD/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 a...

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis

“…Heme auxotrophy was required to facilitate sterol uptake for erg26 mutants. As shown previously (Gachotte et al, 1998;Rahier et al, 2006), the erg26 strain transformed with pVT-At3bHSD/D was capable of growing aerobically without ergosterol supplementation, while erg26 null and erg26/pVT-VOID transformants as well as transformants obtained with nonfunctional 3bHSD/D mutants could grow only on an ergosterol (cholesterol)-supplemented medium. To confirm the authenticity of the complementation assay, the strains were picked from the selection plate and the prototrophic strains were grown in a d-ala-containing liquid medium devoid of sterol and the auxotrophic strains were grown in a cholesterol-containing medium.…”

“…In comparison, the auxotrophic erg26 null strain grown in a cholesterol-containing medium accumulated lanosterol (86%) and small amounts of 4a-carboxy-4b,14-dimethyl-cholest-8,24-dien-3b-ol [4] ( Fig. 3; 14%), as shown previously (Gachotte et al, 1998;Rahier et al, 2006). In the presence of d-ala and cholesterol, the erg26 null strain accumulated 4a-carboxy-4b-methyl-cholest-8,24-dien-3b-ol [3] but no ergosterol, as shown previously (Gachotte et al, 1998).…”

“…In the case of an enzyme that is part of a membrane-bound multienzyme complex, interactions with other components of the complex are probably crucial for optimum enzymatic activity. Thus, 3bHSD/D activity was assayed in the microsomal extracts and the corresponding cytosolic fractions were prepared from the different mutants described below, using the standard assay conditions for recombinant 3bHSD/D (as described in "Materials and Methods" and in Rahier et al, 2006). The immunoblotting analysis indicated that the wild-type 3bHSD/D produced a discrete band with the expected M r in the microsomal extracts (Fig.…”

“…Two distinct families of SMO genes have been identified in Arabidopsis (Arabidopsis thaliana; Darnet and Rahier, 2004), and we recently characterized molecularly and enzymatically two bifunctional 3bHSD/Ds ( Fig. 1) from Arabidopsis (Rahier et al, 2006). These hydroxysteroid-dehydrogenases (HSDs) constitute novel members of the short-chain dehydrogenase/ reductase (SDR) family in plants.…”

“…For both groups, the cofactor binding site is localized in the N-terminal part and the substrate binding site in the C-terminal part. Probably because of its unique bifunctionality and membrane association, 3bHSD/D protein shows low sequence identity with other members of the SDR family and forms a differentiated cluster in phylogenetic analysis (Rahier et al, 2006;Wu et al, 2007). According to a recent classification, 3bHSD/D would be a member of the extended SDR family (Kallberg et al, 2002) for which the number of experimentally solved three-dimensional (3D) structures is lower than for the classical family.…”