Purification and characterization of diacylglycerol acyltransferase from the lipid body fraction of an oleaginous fungus.
Diacylglycerol acyltransferase (DGAT) [EC 2.3.1.20] was purified to apparent homogeneity from the lipid body fraction of an oleaginous fungus, Mortierella ramanniana var. angulispora. The enzyme was solubilized from the lipid body fraction with 0.1% Triton X-100, and purified by subsequent column chromatography on Yellow 86 agarose, Superdex-200, Heparin-Sepharose, second Superdex-200, and second Yellow 86 agarose. The enzyme activity was finally enriched 4,802-fold over that of the starting 1,500X g supernatant. The apparent molecular mass of the enzyme was 53 kDa on SDS polyacrylamide gel electrophoresis. The purified enzyme did not exhibit glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, lipase, transacylase, or acyl-CoA hydrolase activities, although 2-monoolein was acylated with about a half of the enzyme activity toward 1,2-diolein. The purified DGAT depended on exogenous sn-1,2-diolein and oleoyl-CoA, with the highest activity at about 200 and 20 microM, respectively. Purified DGAT utilized a broad range of molecular species of both diacylglycerol and acyl-CoA as substrates. The highest activity was observed with sn-1,2-diolein and lauroyl-CoA. Anionic phospholipids such as phosphatidic acid (PA) activated the purified enzyme, as found for the Triton X-100 extract. Sphingosine dose-dependently inhibited DGAT activity activated by PA and basal activity without PA. These results provide a basis for further studies on the molecular mechanism of triacylglycerol biosynthesis and lipid body formation, in which DGAT plays an important role.
Kamisaka
Y
Y
Applied Microbiology Department, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki. kamisaka@nibh.go.jp
Mishra
S
S
Nakahara
T
T
D000217:Acyltransferases / Q000302:isolation & purification*; D002847:Chromatography, Agarose; D051048:Diacylglycerol O-Acyltransferase; D008055:Lipids / Q000032:analysis*; D009090:Mucorales / Q000201:enzymology*; D013379:Substrate Specificity
D008055:Lipids; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase