title: Homology modeling and site-directed mutagenesis reveal catalytic key amino acids of 3beta-hydroxysteroid-dehydrogenase/C4-decarboxylase from Arabidopsis.
abstract: 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.
authors.affiliation: Institut de Biologie Moléculaire des Plantes, CNRS, Unité Propre de Recherche 2357, 67083 Strasbourg cedex, France. alain.rahier@ibmp-ulp.u-strasbg.fr
authors.forename: Alain
authors.initials: A
authors.lastname: Rahier
authors.forename: Marc
authors.initials: M
authors.lastname: Bergdoll
authors.forename: Geneviève
authors.initials: G
authors.lastname: Génot
authors.forename: Florence
authors.initials: F
authors.lastname: Bouvier
authors.forename: Bilal
authors.initials: B
authors.lastname: Camara
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010840:Phytosterols / Q000737:chemistry; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017384:Sequence Deletion; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D010840:Phytosterols; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; C119754:4alpha-carboxysterol-C3 dehydrogenase; D002262:Carboxy-Lyases