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Title: Membrane topology and identification of key residues of Ea DAcT, a plant MBOAT with unusual substrate specificity

Euonymus alatusdiacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl‐CoA to thesn‐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 thatEaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)‐specific marker. By mapping the membrane topology ofEaDAcT, we obtained an experimentally determined topology model for a plantMBOAT. TheEaDAcT model contains four transmembrane domains (TMDs), with both the N‐ and C‐termini orientated toward the lumen of theER. In addition, there is a large cytoplasmic loop between the first and secondTMDs, with theMBOATsignature region of the protein embedded in the thirdTMDclose 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 fromEuonymus fortuneipossessed the highest activityin vivoandin vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential forEaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity ofEaDAcT, suggesting that multiple amino acids are important for substrate recognition.

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Author(s) / Creator(s):
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Date Published:
Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 82-94
Medium: X
Sponsoring Org:
National Science Foundation
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