Homology Modeling and Site-Directed Mutagenesis Reveal Catalytic Key Amino Acids of 3β-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 3b-hydroxysteroid-dehydrogenase/C4-decarboxylase (3bHSD/D). We recently identified Arabidopsis (Arabidopsis thaliana) 3bHSD/D as a bifunctional short-chain dehydrogenase/reductase protein. We made use of three-dimensional homology modeling to identify key amino acids involved in 4a-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 3bHSD/D. We show that tyrosine-159 and lysine-163, which are oriented near the 3b-hydroxyl group of the substrate in the model, are essential for the 3bHSD/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 4a-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 3bHSD/D for NAD + . Data obtained with serine-133 mutants suggest close proximity between the serine-133 residue and the C4b domain of the bound sterol. Based on these data, we propose a tentative mechanism for 3bHSD/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.

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p

“…The docked substrate and NAD + cofactor interactions with the S. cerevisiae Erg26p active site shown by the model were consistent with those previously determined in the case of the plant A. thaliana 3βHSD/D (ref. 13 ; Fig 3c,f ). These include the conserved catalytic Tyr151xxxLys155 motif proximal to the 3β-hydroxyl group of the carboxysterol, the Arg315 residue proximal to the 4α-carboxylic group of the substrate, and the Asp37 residue bridging the two hydroxyl groups of the adenine ribose of NAD + in accordance with the NAD + versus NADP + cofactor specificity.…”

“…Previous work with A. thaliana 3βHSD/D (NSDHL) indicates that the formation of an Arg326-substrate carboxylate salt bridge is required for productive binding of the substrate in the plant enzyme active site 13 . This is in accordance with the strict substrate specificity of the 3bHSD/D that requires a free C-4 carboxyl group 12 .…”

“…The NSDHL equivalent in S. cerevisiae —Erg26p, encoded by the essential gene ERG26 , also acts in the lanosterol–distal section of the yeast ergosterol synthesis pathway 11 . 3βHSD/D from Arabidopsis thaliana has been characterized and its enzymology has been studied 12 , and essential catalytic and binding residues have been identified 12 13 . According to a homology model and biochemical studies of the A. thaliana enzyme 13 , Tyr159 and Lys163 are oriented near the 3β-hydroxyl group of the substrate and directly involved in the dehydrogenation process, while Arg326 forms an essential salt bridge with the 4α-carboxyl group of the substrate.…”

“…3βHSD/D from Arabidopsis thaliana has been characterized and its enzymology has been studied 12 , and essential catalytic and binding residues have been identified 12 13 . According to a homology model and biochemical studies of the A. thaliana enzyme 13 , Tyr159 and Lys163 are oriented near the 3β-hydroxyl group of the substrate and directly involved in the dehydrogenation process, while Arg326 forms an essential salt bridge with the 4α-carboxyl group of the substrate. The Asp39 residue is thought to contact the hydroxyl groups of the adenosine-ribose ring of NAD + .…”

The odd one out:Arabidopsisreticulon20 has a role in lipid biosynthesis

“…One of the recently characterized 3BETAHSD/D2 proteins (Rahier et al , 2006; 3BETAHSD/D2) is actually a reticulon protein (RTN19). By using a three-dimensional homology modeling to identify key amino acids, it has been determined that this protein is a bifunctional short-chain dehydrogenase/reductase enzyme (Rahier et al, 2009; Rahier et al, 2013).…”