Expression and characterization of diacylglycerol acyltransferase from Arabidopsis thaliana in insect cell cultures.
Diacylglycerol acyltransferase (DGAT) catalyses the acylation of the sn-3 hydroxy group of sn-1,2-diacylglycerol using acyl-CoA. The gene encoding DGAT from Arabidopsis thaliana has been cloned and the function of the enzyme proved by expression of the coding sequence using a bacculovirus expression system in insect cell cultures. The expressed protein catalysed the synthesis of [(14)C]triacylglycerol from [(14)C]diacylglycerol and oleoyl-CoA. The heterologously expressed DGAT activity was found mostly associated with the 100000 g pellet. The optimum activity was achieved at a neutral pH, in the presence of Mg2+, and at an optimum oleoyl-CoA concentration of 20 microM. The DGAT used the substrates palmitoyl-CoA and oleoyl-CoA equally effectively. In these experiments, the inclusion of recombinant acyl-CoA binding protein had a relatively small effect upon DGAT activity.
Hobbs
D H
DH
John Innes Centre, Norwich Research Park, Colney, Norwich, Norfolk NR4 7UH, UK.
Hills
M J
MJ
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D016367:Baculoviridae; D002460:Cell Line; D051048:Diacylglycerol O-Acyltransferase; D004075:Diglycerides / Q000378:metabolism; D007313:Insecta; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D000214:Acyl Coenzyme A; D004075:Diglycerides; D011994:Recombinant Proteins; C017585:oleoyl-coenzyme A; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase; C013965:diolein