Publications

• Homology modeling and site-directed mutagenesis reveal catalytic key amino acids of 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase from Arabidopsis.

  Rahier A., Bergdoll M., Genot G., Bouvier F., Camara B.

  Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzyme complex including a 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase (3betaHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3betaHSD/D as a bifunctional short-chain dehyd ... >> More

  Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzyme complex including a 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase (3betaHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3betaHSD/D as a bifunctional short-chain dehydrogenase/reductase protein. We made use of three-dimensional homology modeling to identify key amino acids involved in 4alpha-carboxy-sterol and NAD binding and catalysis. Key amino acids were subjected to site-directed mutagenesis, and the mutated enzymes were expressed and assayed both in vivo and in vitro in an erg26 yeast strain defective in 3betaHSD/D. We show that tyrosine-159 and lysine-163, which are oriented near the 3beta-hydroxyl group of the substrate in the model, are essential for the 3betaHSD/D activity, consistent with their involvement in the initial dehydrogenation step of the reaction. The essential arginine-326 residue is predicted to form a salt bridge with the 4alpha-carboxyl group of the substrate, suggesting its involvement both in substrate binding and in the decarboxylation step. The essential aspartic acid-39 residue is in close contact with the hydroxyl groups of the adenosine-ribose ring of NAD+, in good agreement with the strong preference of 3betaHSD/D for NAD+. Data obtained with serine-133 mutants suggest close proximity between the serine-133 residue and the C4beta domain of the bound sterol. Based on these data, we propose a tentative mechanism for 3betaHSD/D activity. This study provides, to our knowledge, the first data on the three-dimensional molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthesis pathway with its substrate. The implications of our findings for studying the roles of C4-alkylated sterol precursors in plant development are discussed. << Less

  Plant Physiol. 149:1872-1886(2009) [PubMed] [EuropePMC]

• Molecular and enzymatic characterizations of novel bifunctional 3beta-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana.

  Rahier A., Darnet S., Bouvier F., Camara B., Bard M.

  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 ... >> More

  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 4alpha-carboxy-cholest-7-en-3beta-ol and its 3alpha-deuterated analog revealed that 3alpha-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-5alpha-ergosta-9beta,19-cyclo-24(24(1))-en-4alpha-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. << Less

  J. Biol. Chem. 281:27264-27277(2006) [PubMed] [EuropePMC]