Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

Euonymus alatus diacylglycerol acetyltransferase ( Ea DAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylgylycerols (acetyl-TAGs) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, Ea DAcT transfers the acetyl group from acetyl-CoA to sn -1,2 diacylglycerol (DAG) to produce acetyl-TAGs. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared to acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by Ea DAcT. This extreme substrate specificity of Ea DAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that Ea DAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, Ea DAcT could only acetylate the free hydroxyl group of sn -1,2 DAG but not the available hydroxyl groups in sn -1,3 DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, Ea DAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, Ea DAcT was capable of synthesizing alkyl acetates although the efficiency of production was low. This improved understanding of Ea DAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn -1,2 DAGs and will be helpful in engineering the production of acetyl-TAGs with improved functionality in transgenic plants.