Received November 13, 2008
Accepted February 11, 2009
Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3
-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis thaliana
Alain Rahier *, Marc Bergdoll , Genevieve Genot , Florence Bouvier , and Bilal Camara
From the Institut de Biologie Moleculaire des Plantes, CNRS, UPR2357, 28 rue Goethe, 67083 Strasbourg cedex, France
* Corresponding author; email: alain.rahier{at}ibmp-ulp.u-strasbg.fr.
Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzymatic complex including a 3
-hydroxysteroid-dehydrogenase/C4-decarboxylase (3HSD/D). We recently identified A.thaliana 3HSD/D as a bifunctional short-chain dehydrogenase/reductase (SDR) protein. We made use of three dimensional homology modeling to identify key amino-acids involved in 4
-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 3HSD/D. We show that Tyr159 and Lys163 which are oriented near the 3-hydroxyl group of the substrate in the model, are essential for the 3HSD/D activity, consistent with their involvement in the initial dehydrogenation step of the reaction. The essential Arg326 residue is predicted to form a salt bridge with the 4-carboxyl group of the substrate suggesting its involvement both in substrate binding and in the decarboxylation step. The essential Asp39 residue is in close contact with the hydroxyl groups of the adenosine-ribose ring of NAD+ in good agreement with the strong preference of 3HSD/D for NAD+. Data obtained with Ser133 mutants suggest close proximity between Ser133 residue and the C4-domain of the bound sterol. Based on the data, we propose a tentative mechanism for 3HSD/D activity. This study provides the first data on the three-dimensional molecular interactions of an enzyme of the post oxido-squalene 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.
