Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity

Abstract

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) 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). EaDAcT 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 EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT 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 EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…The important motifs are shown in the topology including the MBOAT signature region with putative active site residues, and the ER retrieval motif. This figure was developed based on information from Tran et al (). (d) Molecular model of Arabidopsis PDAT using phospholipase A 2 as template and PHYRE2 protein fold recognition server.…”

2‐Acetyl‐1,3‐Diacyl–sn‐Glycerols with Unusual Acyl Composition in Seed Oils of the Genus Polygala

“…While TAGs are the most abundant lipid in the fleshy aril (92%), sn‐ 3‐acTAGs dominate the profile of the seed endosperm and embryo at 95 and 66% of total lipids, respectively . Biosynthesis has been demonstrated to be dependent on a divergent member of the membrane‐bound O‐acyltransferase (MBOAT) protein family referred to as E. alatus diacylglycerol acetyltransferase ( Ea DAcT), an enzyme capable of acetylating the sn‐ 3 position of diacylglycerol (DAG) . sn‐ 3‐acTAGs isolated from E. alatus seeds have lower kinematic viscosity than typical TAG oils, generating interest as a potential biofuel and triggering the genetic engineering of camelina ( Camelina sativa) and soybean ( Glycine max ) as novel sn‐ 3‐acTAG production platforms .…”

“…Biosynthesis has been demonstrated to be dependent on a divergent member of the membrane‐bound O‐acyltransferase (MBOAT) protein family referred to as E. alatus diacylglycerol acetyltransferase ( Ea DAcT), an enzyme capable of acetylating the sn‐ 3 position of diacylglycerol (DAG) . sn‐ 3‐acTAGs isolated from E. alatus seeds have lower kinematic viscosity than typical TAG oils, generating interest as a potential biofuel and triggering the genetic engineering of camelina ( Camelina sativa) and soybean ( Glycine max ) as novel sn‐ 3‐acTAG production platforms . The lower calorific value of sn‐ 3‐acTAGs also suggests possible applications in the reduced‐calorie food markets …”

A New Class of Acetyl‐TAG Present in Seed Oils of Polygala Species

“…The most extreme example of chain length variation however, is exemplified by some plant species that produce TAG with acetic acid esterified to the glycerol backbone, usually at the sn ‐3 position. Most of the species that are known to synthesize these unusual 3‐acetyl‐1,2‐diacylglycerols (acetyl‐TAG) are members of the Celastreae family, including many different Euonymus species . The ability to produce acetyl‐TAG also extends beyond the Celastreae family, with acetyl‐TAG also being found in the seeds of a few members of the Lardizabalaceae, Ranunculaceae, Rosaceae, and Balsaminaceae families .…”

“…Most of the species that are known to synthesize these unusual 3‐acetyl‐1,2‐diacylglycerols (acetyl‐TAG) are members of the Celastreae family, including many different Euonymus species . The ability to produce acetyl‐TAG also extends beyond the Celastreae family, with acetyl‐TAG also being found in the seeds of a few members of the Lardizabalaceae, Ranunculaceae, Rosaceae, and Balsaminaceae families . Further, acetyl‐TAGs are not just restricted to the plant kingdom, as these unusual storage lipid molecules have also been detected in bovine udders and in the fat body cells of the goldenrod gall fly, Eurosta solidaginis …”