Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape.
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 (22:1cisDelta13)-CoA over oleoyl (18:1cisDelta9)-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.


CONCLUSIONS
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.
Weselake
Randall J
RJ
Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada. randall.weselake@ualberta.ca
Madhavji
Milan
M
Szarka
Steve J
SJ
Patterson
Nii A
NA
Wiehler
William B
WB
Nykiforuk
Cory L
CL
Burton
Tracy L
TL
Boora
Parveen S
PS
Mosimann
Steven C
SC
Foroud
Nora A
NA
Thibault
Benjamin J
BJ
Moloney
Maurice M
MM
Laroche
André
A
Furukawa-Stoffer
Tara L
TL
D000214:Acyl Coenzyme A / Q000378:metabolism; D029688:Brassica napus / Q000201:enzymology*; D018076:DNA, Complementary; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D051048:Diacylglycerol O-Acyltransferase