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Permanent link (DOI): https://doi.org/10.7939/R3GM82262
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Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape Open Access
- Author or creator
Weselake, Randall J.
Szarka, Steve J.
Patterson, Nii A.
Wiehler, William B.
Nykiforuk, Cory L.
Burton, Tracy L.
Boora, Parveen S.
Mosimann, Steven C.
Foroud, Nora A.
Thibault, Benjamin J.
Moloney, Maurice M.
Furukawa-Stoffer, Tara L.
- Additional contributors
Recombinant Proteins/Isolation & Purification
Acyl Coenzyme A/Metabolism
Plant Proteins/Isolation & Purification
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Diacylglycerol O-Acyltransferase/Isolation & Purification
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Background Diacylglycerol acyltransferase (DGAT, EC 220.127.116.11) 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.
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Weselake, R. J., Madhavji, M., Szarka, S. J., Patterson, N. A., Wiehler, W. B., Nykiforuk, C. L., Burton, T. L., Boora, P. S., Mosimann, S. C., Foroud, N. A., Thibault, B. J., Moloney, M. M., Laroche, A., & Furukawa-Stoffer, T. L. (2006). Acyl-CoA-binding and self-associating properties of a recombinant 13.3 kDa N-terminal fragment of diacylglycerol acyltransferase-1 from oilseed rape. BMC Biochemistry, 7(24), [13 pages]. http://dx.doi.org/10.1186/1471-2091-7-24
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