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

• Author(s) / Creator(s)

  • Nykiforuk, Cory L.
  • Szarka, Steve J.
  • Laroche, André
  • Mosimann, Steven C.
  • Burton, Tracy L.
  • Thibault, Benjamin J.
  • Boora, Parveen S.
  • Madhavji, Milan
  • Foroud, Nora A.
  • Moloney, Maurice M.
  • Wiehler, William B.
  • Patterson, Nii A.
  • Weselake, Randall J.
  • Furukawa-Stoffer, Tara L.

• 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 (22:1cisΔ13)-CoA over oleoyl (18:1cisΔ9)-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.

• Date created

  2006

• Subjects / Keywords

  • Plant Proteins/Chemistry
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Recombinant Proteins/Metabolism
  • Diacylglycerol O-Acyltransferase/Chemistry
  • Diacylglycerol O-Acyltransferase/Isolation & Purification
  • Diacylglycerol O-Acyltransferase/Genetics
  • Acyl Coenzyme A/Metabolism
  • Plant Proteins/Genetics
  • Dna, Complementary
  • Recombinant Proteins/Isolation & Purification
  • Diacylglycerol O-Acyltransferase/Metabolism
  • Plant Proteins/Metabolism
  • Structure-Activity Relationship
  • Plant Proteins/Isolation & Purification
  • Recombinant Proteins/Genetics
  • Substrate Specificity
  • Brassica Napus/Enzymology
  • Recombinant Proteins/Chemistry

• Type of Item

  Article (Published)

• DOI

  https://doi.org/10.7939/R3GM82262

• License

  Attribution 4.0 International