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

Abstract

Euonymus alatus diacylglycerol 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-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. 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 with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT 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 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, EaDAcT 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, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT 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-TAG with improved functionality in transgenic plants.

Keywords: MBOAT; acetyl-TAGs; acetyltransferase; substrate specificity; triacylglycerols.

Figure 1. EaDAcT utilizes very short-chain acyl-CoA molecules

(A) Yeast microsomes expressing EaDAcT were incubated with 250 μM of acyl-CoA with different acyl chain lengths. Reaction products were detected using ESI-MS scans for the neutral loss of the sn-3 acyl group and normalized to a 3-acyl-1,2-dipentadecanoyl-sn-glycerol standard that possessed the same sn-3 acyl group. Duplicate reactions were performed for each acyl-CoA substrate. These results are representative of at least three independent experiments performed on microsomes isolated at different times. (B) The acyltransferase activity of EaDAcT using different short chain acyl-CoA substrates was determined across a range of substrate concentrations. Reaction products were quantified using ESI-MS scans for the neutral loss of the sn-3 acyl group. Values are expressed as mean ± S.D. for three independent assays. Curves were fitted using non-linear regression (GraphPad Prism).

Figure 2. EaDAcT preferentially acetylates more unsaturated DAG molecular species in vitro

(A) Quantification of DAG molecular species initially present in yeast microsomes expressing EaDAcT. Values represent the mean ± S.D. for three independent lipid extractions. (B) Quantification of acetyl-TAG molecular species formed after incubation of the same EaDAcT microsomes with different concentrations of acetyl-CoA. Values represent the mean ± S.D. for three independent assays. For both DAG (A) and acetyl-TAG (B) molecular species, the number of acyl carbons (x) and double bonds (y) is indicated by x:y. Asterisks indicate statistical differences between the DAG or acetyl-TAG molecular species containing the same number of acyl carbons but different numbers of double bonds (unpaired t-test; *, P<0.05; **, P<0.01).

Figure 3. EaDAcT can acetylate DAG containing different chain length fatty acids

Positive ESI mass spectra obtained from the neutral loss of ammonium acetate from lipid products resulting from incubating microsomes containing EaDAcT with acetyl-CoA and different exogenous DAG substrates. Peaks correspond to m/z values of the [M+NH₄]⁺ adduct of the intact acetyl-TAG molecule. The number of acyl carbons in each series of acetyl-TAG molecules is indicated; for clarity, the number of double bonds (x) is not defined.

Figure 4. EaDAcT can acetylate DAG containing an sn-2 acetyl-group

Positive ESI mass spectra obtained from the neutral loss of ammonium acetate from lipid products resulting from incubating microsomes containing EaDAcT with acetyl-CoA and 1-oleoyl-2-acetyl-sn-glycerol. Peaks correspond to m/z values of the [M+NH₄]⁺ adduct of the intact acetyl-TAG molecule. The number of acyl carbons in each series of acetyl-TAG molecules is indicated; for clarity, the number of double bonds (x) is not defined.

Figure 5. EaDAcT can acetylate a range of fatty alcohols in vitro

TLC separation of total lipids extracted yeast microsomes expressing EaDAcT incubated with [1-¹⁴C] acetyl-CoA and 125 μM of different chain length alcohols. Fatty alcohols are denoted using x:y where x indicates the number of carbons and y the number of double bonds.

Figure 6. EaDAcT can acetylate fatty alcohols in vivo

(A) GC–MS chromatograms of alkyl acetates purified from H1246 yeast expressing the empty vector pESC-URA or combinations of AmFAR1 and EaDAcT. (B) Electron impact mass spectrum of the stearyl acetate peak (retention time=13.1 min) labelled with diagnostic fragment ions. (C) Quantification of alkyl acetates from (A). Values represent mean ± S.D. for three biological replicates.