Acyl CoA:diacylgycerol acyltransferase (EC 2.3.1.20; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungus Mortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes as MrDGAT2A and MrDGAT2B. Diacylglycerol acyltransferase (DGAT)1 is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi, and mammals (for reviews, see Refs. 1-3). The enzyme is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (DAG) to form triacylglycerol (TAG). As the final step in TAG biosynthesis via the Kennedy pathway, it is the only step not involved in membrane biosynthesis. In plants and fungi, DGAT is associated with the membrane and lipid body fractions, particularly in oilseeds, where it contributes to the storage of carbon used as energy reserves. In animals, the role of DGAT is more complex. Triacylglycerols are synthesized and stored in several cell types including adipocytes and hepatocytes (4), but, in addition, DGAT may play a role in lipoprotein assembly and the regulation of plasma triacylglycerol concentration (4), as well as participate in the regulation of DAG levels (5, 6).Cases et al. (7) reported the first cloning of a DGAT gene from mouse. Using coding sequences from acyl CoA:cholesterol acyltransferase (ACAT), expressed sequence tag data bases were searched and a gene identified that shared 20% identity with the mouse ACAT. After cloning and expression of the gene in insect cells, no ACAT activity was detected in isolated membranes; however, using [1-14 C]oleoyl-CoA as substrate, a range of acceptors was examined and Cases et al. discovered DAG was the acceptor molecule, thus demonstrating DGAT activity. Hobbs et al. (8) reported the cloning of an Arabidopsis homologue of the mouse DGAT gene and confirmed the presence of DGAT activity in insect cells expressing the cDNA. Southern ...
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