Defining the extreme substrate specificity of diacylglycerol acetyltransferase, an unusual membrane-bound -acyltransferase.

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