Research article

Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape

Randall J Weselake¹ , Milan Madhavji² , Steve J Szarka^3,5 , Nii A Patterson^3,6 , William B Wiehler² , Cory L Nykiforuk^2,5 , Tracy L Burton² , Parveen S Boora² , Steven C Mosimann² , Nora A Foroud² , Benjamin J Thibault² , Maurice M Moloney³ , André Laroche⁴ and Tara L Furukawa-Stoffer²

¹Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada

²Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada

³Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada

⁴Lethbridge Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta P.O. Box 3000 Main, Lethbridge, Alberta, T1J 4B1, Canada

⁵Present address : SemBioSys Genetics Inc., 110, 2985 23 Avenue N.E., Calgary, AB T1Y 7L3, Canada

⁶Present address: Metabolix Inc., 21 Erie Street, Cambridge, MA 02139, USA

author email corresponding author email

BMC Biochemistry 2006, 7:24doi:10.1186/1471-2091-7-24

Published:	27 December 2006

Abstract

Background

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1_(1–116)His₆, with calculated molecular mass of 13,278 Da.

Results

BnDGAT1_(1–116)His₆ was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1_(1–116)His₆ interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cis^Δ13)-CoA over oleoyl (18:1cis^Δ9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1_(1–116)His₆ self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1_(1–116)His₆ failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER.

Conclusion

Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER.