Defining the extreme substrate specificity ofEuonymus alatusdiacylglycerol acetyltransferase, an unusual membrane-boundO-acyltransferase.
Euonymus alatusdiacylglycerol acetyltransferase (EaDAcT) 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-boundO-acyltransferase (MBOAT) family,EaDAcT transfers the acetyl group from acetyl-CoA tosn-1,2-diacylglycerol (DAG) to produce acetyl-TAG.In vitroassays 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 byEaDAcT. This extreme substrate specificity ofEaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups.In vitroselectivity experiments revealed thatEaDAcT 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,EaDAcT could only acetylate the free hydroxyl group ofsn-1,2-DAG but not the available hydroxyl groups insn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase,EaDAcT could acetylate fatty alcoholsin vitroto produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols,EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding ofEaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylatesn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, U.S.A.
Sunil
S
Bansal
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, U.S.A. tdurrett@ksu.edu.
Timothy P
TP
Durrett
MBOAT; acetyl-TAGs; acetyltransferase; substrate specificity; triacylglycerols
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