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PLoS One ; 11(10): e0165431, 2016.
Article in English | MEDLINE | ID: mdl-27780240

ABSTRACT

The membrane proteins acyl-CoA:diacylglycerol acyltransferases (DGAT) are essential actors for triglycerides (TG) biosynthesis in eukaryotic organisms. Microbial production of TG is of interest for producing biofuel and value-added novel oils. In the oleaginous yeast Yarrowia lipolytica, Dga1p enzyme from the DGAT2 family plays a major role in TG biosynthesis. Producing recombinant DGAT enzymes pure and catalytically active is difficult, hampering their detailed functional characterization. In this report, we expressed in Escherichia coli and purified two soluble and active forms of Y. lipolytica Dga1p as fusion proteins: the first one lacking the N-terminal hydrophilic segment (Dga1pΔ19), the second one also devoid of the N-terminal putative transmembrane domain (Dga1pΔ85). Most DGAT assays are performed on membrane fractions or microsomes, using radiolabeled substrates. We implemented a fluorescent assay in order to decipher the substrate specificity of purified Dga1p enzymes. Both enzyme versions prefer acyl-CoA saturated substrates to unsaturated ones. Dga1pΔ85 preferentially uses long-chain saturated substrates. Dga1p activities are inhibited by niacin, a specific DGAT2 inhibitor. The N-terminal transmembrane domain appears important, but not essential, for TG biosynthesis. The soluble and active proteins described here could be useful tools for future functional and structural studies in order to better understand and optimize DGAT enzymes for biotechnological applications.


Subject(s)
Diacylglycerol O-Acyltransferase/metabolism , Fungal Proteins/metabolism , Yarrowia/enzymology , Amino Acid Sequence , Diacylglycerol O-Acyltransferase/antagonists & inhibitors , Diacylglycerol O-Acyltransferase/genetics , Fatty Acids/metabolism , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/genetics , Molecular Sequence Data , Niacin/chemistry , Niacin/metabolism , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Sequence Alignment , Substrate Specificity , Triglycerides/metabolism
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