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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-Hydroxysteroid-Dehydrogenase/C4-Decarboxylase from Arabidopsis^[C],[W]

Institut de Biologie Moléculaire des Plantes, CNRS, Unité Propre de Recherche 2357, 67083 Strasbourg cedex, France

Sterols become functional only after removal of the two methyl groups at C4 by a membrane-bound multienzyme complex including a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3βHSD/D as a bifunctional short-chain dehydrogenase/reductase 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 3βHSD/D. We show that tyrosine-159 and lysine-163, which are oriented near the 3β-hydroxyl group of the substrate in the model, are essential for the 3βHSD/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 4-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 3βHSD/D for NAD⁺. Data obtained with serine-133 mutants suggest close proximity between the serine-133 residue and the C4β domain of the bound sterol. Based on these data, we propose a tentative mechanism for 3βHSD/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.

^[C] Some figures in this article are displayed in color online but in black and white in the print edition.

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.132282

^* Corresponding author; e-mail alain.rahier{at}ibmp-ulp.u-strasbg.fr.

Received November 13, 2008; accepted February 11, 2009; published February 13, 2009.