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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.

Weselake RJ, Madhavji M, Szarka SJ, Patterson NA, Wiehler WB, Nykiforuk CL, Burton TL, Boora PS, Mosimann SC, Foroud NA, Thibault BJ, Moloney MM, Laroche A, Furukawa-Stoffer TL - BMC Biochem. (2006)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada. randall.weselake@ualberta.ca

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 (22:1cisDelta13)-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.

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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Analysis of self-associating properties of BnDGAT1(1–116)His6 using gel filtration chromatography and chemical cross-linking. Panel A: Gel filtration chromatography of clarified lysate of bacteria overexpressing BnDGAT1(1–116)His6 E. coli producing BnDGAT1(1–116)His6 and SDS-PAGE of protein in fractions eluted from the column. IPTG-induced bacteria were resuspended in 30 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The Superdex 75 HR 10/30 column was equilibrated with extraction buffer (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) and operated at room temperature with a flow rate of 0.5 mL/min using an FPLC system. One hundred microliters of extract were applied to the column and 1 ml fractions were collected during elution. Ten microliter aliquots were analyzed by SDS-PAGE using a separating gel prepared with a concentration of 15% monomer and 1.1% cross-linker. The column and SDS gel were calibrated with molecular mass markers. Panel B: Chemical cross-linking of BnDGAT1(1–116)His6 monitored by Western blotting of BnDGAT1(1–116)His6 before (lane 1) and after (lane 2) treatment with DMS. BnDGAT1(1–116)His6 (500 μg/mL) was cross-linked in the presence of 4 mg DMS per ml in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to application of 10 μL samples to an SDS-PAGE gel, which was prepared using a concentration of 10% total monomer and 1.1% cross-linker.
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Figure 4: Analysis of self-associating properties of BnDGAT1(1–116)His6 using gel filtration chromatography and chemical cross-linking. Panel A: Gel filtration chromatography of clarified lysate of bacteria overexpressing BnDGAT1(1–116)His6 E. coli producing BnDGAT1(1–116)His6 and SDS-PAGE of protein in fractions eluted from the column. IPTG-induced bacteria were resuspended in 30 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The Superdex 75 HR 10/30 column was equilibrated with extraction buffer (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) and operated at room temperature with a flow rate of 0.5 mL/min using an FPLC system. One hundred microliters of extract were applied to the column and 1 ml fractions were collected during elution. Ten microliter aliquots were analyzed by SDS-PAGE using a separating gel prepared with a concentration of 15% monomer and 1.1% cross-linker. The column and SDS gel were calibrated with molecular mass markers. Panel B: Chemical cross-linking of BnDGAT1(1–116)His6 monitored by Western blotting of BnDGAT1(1–116)His6 before (lane 1) and after (lane 2) treatment with DMS. BnDGAT1(1–116)His6 (500 μg/mL) was cross-linked in the presence of 4 mg DMS per ml in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to application of 10 μL samples to an SDS-PAGE gel, which was prepared using a concentration of 10% total monomer and 1.1% cross-linker.

Mentions: Analysis of the molecular mass of BnDGAT1(1–116)His6 under non-denaturing conditions was conducted using gel filtration chromatography of clarified lysate of ruptured bacteria expressing the N-terminal fragment (Figure 4A). Analysis of protein in the fractions eluting from the column by SDS-PAGE indicated that BnDGAT1(1–116)His6 eluted between the elution positions of carbonic anhydrase (29 kDa) and BSA (66 kDa).


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

Weselake RJ, Madhavji M, Szarka SJ, Patterson NA, Wiehler WB, Nykiforuk CL, Burton TL, Boora PS, Mosimann SC, Foroud NA, Thibault BJ, Moloney MM, Laroche A, Furukawa-Stoffer TL - BMC Biochem. (2006)

Analysis of self-associating properties of BnDGAT1(1–116)His6 using gel filtration chromatography and chemical cross-linking. Panel A: Gel filtration chromatography of clarified lysate of bacteria overexpressing BnDGAT1(1–116)His6 E. coli producing BnDGAT1(1–116)His6 and SDS-PAGE of protein in fractions eluted from the column. IPTG-induced bacteria were resuspended in 30 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The Superdex 75 HR 10/30 column was equilibrated with extraction buffer (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) and operated at room temperature with a flow rate of 0.5 mL/min using an FPLC system. One hundred microliters of extract were applied to the column and 1 ml fractions were collected during elution. Ten microliter aliquots were analyzed by SDS-PAGE using a separating gel prepared with a concentration of 15% monomer and 1.1% cross-linker. The column and SDS gel were calibrated with molecular mass markers. Panel B: Chemical cross-linking of BnDGAT1(1–116)His6 monitored by Western blotting of BnDGAT1(1–116)His6 before (lane 1) and after (lane 2) treatment with DMS. BnDGAT1(1–116)His6 (500 μg/mL) was cross-linked in the presence of 4 mg DMS per ml in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to application of 10 μL samples to an SDS-PAGE gel, which was prepared using a concentration of 10% total monomer and 1.1% cross-linker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1764880&req=5

Figure 4: Analysis of self-associating properties of BnDGAT1(1–116)His6 using gel filtration chromatography and chemical cross-linking. Panel A: Gel filtration chromatography of clarified lysate of bacteria overexpressing BnDGAT1(1–116)His6 E. coli producing BnDGAT1(1–116)His6 and SDS-PAGE of protein in fractions eluted from the column. IPTG-induced bacteria were resuspended in 30 ml of 100 mM sodium phosphate buffer (pH 7.4) containing 150 mM NaCl. The suspension was passed through a French press three times at 20,000 p.s.i.. The suspension of ruptured bacteria was centrifuged at 13,000 g for 15 min to obtain a clarified lysate. The Superdex 75 HR 10/30 column was equilibrated with extraction buffer (100 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) and operated at room temperature with a flow rate of 0.5 mL/min using an FPLC system. One hundred microliters of extract were applied to the column and 1 ml fractions were collected during elution. Ten microliter aliquots were analyzed by SDS-PAGE using a separating gel prepared with a concentration of 15% monomer and 1.1% cross-linker. The column and SDS gel were calibrated with molecular mass markers. Panel B: Chemical cross-linking of BnDGAT1(1–116)His6 monitored by Western blotting of BnDGAT1(1–116)His6 before (lane 1) and after (lane 2) treatment with DMS. BnDGAT1(1–116)His6 (500 μg/mL) was cross-linked in the presence of 4 mg DMS per ml in 0.2 M triethanolamine-HCl at pH 8.5. Cross-linking reactions were allowed to proceed at room temperature for 3 h. The reactions were quenched with 2 × SDS loading buffer and boiled for 5 min prior to application of 10 μL samples to an SDS-PAGE gel, which was prepared using a concentration of 10% total monomer and 1.1% cross-linker.
Mentions: Analysis of the molecular mass of BnDGAT1(1–116)His6 under non-denaturing conditions was conducted using gel filtration chromatography of clarified lysate of ruptured bacteria expressing the N-terminal fragment (Figure 4A). Analysis of protein in the fractions eluting from the column by SDS-PAGE indicated that BnDGAT1(1–116)His6 eluted between the elution positions of carbonic anhydrase (29 kDa) and BSA (66 kDa).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agricultural, Food and Nutritional Science, 4-10 Agriculture/Forestry Centre, University of Alberta, Edmonton, Alberta, T6G 2P5, Canada. randall.weselake@ualberta.ca

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 (22:1cisDelta13)-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.

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.

Show MeSH
Related in: MedlinePlus