Identification and functional analysis of the phospholipid:diacylglycerol acyltransferase, a putative enzyme involved in triacylglycerol synthesis in Chlamydomonas reinhardtii
Kangsup Yoon, Danxiang Han, Yantao Li, Milton Sommerfeld and Qiang Hu
Laboratory for Algae Research and Biotechnology, Arizona State University, Mesa AZ 85212
A full-length cDNA encoding a putative phospholipid:diacylglycerol acyltransferase was obtained from the green microalga Chlamydomonas reinhardtii. A 3123 bp open reading frame of this cDNA, designated as CrPDAT, encodes a protein of 1041 amino acids that shows moderate sequence homology to PDATs, PSATs and LCATs from diverse organisms. A partial CrPDAT gene, lacking a predicted membrane spanning region was fused in frame with alpha-factor secretion signal and functionally expressed in the yeast Pichia pastoris under the control of the methanol inducible alcohol oxidase promoter. In vitro enzymatic activity assays revealed that the resulting recombinant CrPDAT protein ( ΔTMCrPDAT) has multiple catalytic functions: 1) transacylation of diacylglycerol (DAG) with acyl groups from various phospholipids (PL) to form triacylglycerol (TAG); 2) DAG:DAG transacylation to form TAG and monoacylglycerol; and 3) functioning as a lipase to hydrolyze DAG, TAG and PL into free fatty acids. Among a number of PL species tested, phosphatidylinositol was the most preferred substrate for ΔTMCrPDAT. ΔTMCrPDAT exhibited a higher affinity to 1,2-DAG than to 1,3-DAG as an acyl acceptor, demonstrating that the rate of activity is highly dependent on acyl position towards TAG synthesis. Transcriptional expression of CrPDAT and shortgun mass spectrometry-based glycerolipids profiling of Chlamydomonas cells under TAG induction conditions indicated that CrPDAT contributes to TAG production under high light and nitrogen starvation conditions. In vivo functional analysis of CrPDAT and the subcellular localization of CrPDAT in Chlamydomonas cells are underway. The strong lipase activity of ΔTMCrPDAT, which has not been reported for the PDATs of other organisms, suggests that this enzyme could be a candidate as a biocatalyst for lipid hydrolysis and conversion.
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