Abstracts

Structuralfeatures and functional residues important for the activity of anunusual membrane bound O-acyltransferase

by Tam Nguyen Tran




Institution: Kansas State University
Department:
Year: 2017
Keywords: acetyl-TAG; MBOAT; Membranetopology; Euonymusalatus diacylglycerol acetyltransferase; Waxsynthase
Posted: 02/01/2018
Record ID: 2182150
Full text PDF: http://hdl.handle.net/2097/35494


Abstract

The membrane bound O-acyltransferase (MBOAT) familycontains multi-pass membrane proteins that add fatty acids todifferent compounds. Despite their importance in economic activityand human health, little is known about the localization of theactive site and regions important for determining substratespecificity of MBOATs. Euonymus alatus diacylglycerolacetyltransferase (EaDAcT) is the only known MBOAT enzyme thatexhibits a high preference for acetyl-CoA, the shortest possibleacyl-CoA. EaDAcT catalyzes the transfer of the acetate group fromacetyl-CoA to the sn-3 position of diacylglycerol to form3-acetyl-1,2-diacyl-sn-glycerol. Our goal was to investigate thestructural features and the amino acid residues that definesubstrate specificity of EaDAcT to provide insights into themechanism by which MBOAT family controls substrate selection. Bymapping the membrane topology of EaDAcT we obtained the firstexperimentally determined topology model for a plant MBOAT. TheEaDAcT model contains four transmembrane domains with both the N-and C- termini oriented toward the endoplasmic reticulum lumen. TheMBOAT signature region including the putative active site His-257of the protein is embedded in the third transmembrane domain closeto the interface between the membrane and the cytoplasm. In orderto identify amino acid residues important for acetyltransferaseactivity, we isolated and characterized orthologs of EaDAcT fromother acetyl-TAG producing plants. Among them, theacetyltransferase from Euonymus fortunei possessed the highestactivity in vivo and in vitro. Mutagenesis of conserved residues ofDAcTs revealed that Ser-253, His-257 and Asp-258 are essential forenzyme activity of EaDAcT, suggesting their involvement in theenzyme catalysis. Alteration of residues unique toacetyltransferases did not alter the acyl donor specificity ofEaDAcT, implying that multiple amino acids are important forsubstrate recognition. Together, this work identifies thestructural features of EaDAcT and offers an initial view of theamino acids important for activity of the enzyme.Advisors/Committee Members: Timothy P. Durrett.