Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity. (21st August 2017)

• Record Type: Journal Article
• Authors: Tran, Tam N. T.; Shelton, Jennifer; Brown, Susan; Durrett, Timothy P.
• Is Part Of: Plant journal. Volume 92:Number 1(2017)
• Page Start: 82
• Page End: 94
• Publication Date: 2017-08-21
• Subjects: Euonymus alatus diacylglycerol acetyltransferase -- MBOAT -- membrane topology -- acetyl‐TAG; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.13636
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: Euonymus alatus diacylglycerol acetyltransferase ( Ea DAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to the sn ‐3 position of diacylglycerol to form 3‐acetyl‐1, 2‐diacyl‐ sn ‐glycerol (acetyl‐TAG). Ea DAcT belongs to a small, plant‐specific subfamily of the membrane bound O‐acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that Ea DAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology of Ea DAcT, we obtained an experimentally determined topology model for a plant MBOAT. The Ea DAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl‐TAG‐producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro . Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for Ea DAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of Ea DAcT, suggesting that multiple amino acids are important for substrate recognition. Significance Statement: Unlike other MBOATs, Euonymus alatus diacylglycerol acetyltransferase ( Ea DAcT) uses acetyl‐CoA as its acyl donor substrate to acetylate diacylglycerol, synthesizing unusual acetyl‐triacylglycerols. We mapped the membrane topology of Ea DAcT to obtain an experimentally verified topology model for a plant MBOAT. In order to better understand the unique substrate specificity of the enzyme, we isolated additional acetyltransferases from other plant species that synthesize acetyl‐triacylglycerols, allowing identification of conserved residues important for acetyltransferase activity.