A simple and reproducible non-radiolabeled in vitro assay for recombinant acyltransferases involved in triacylglycerol biosynthesis

Abstract

Diacylglycerol acyltransferase (DGAT) is considered as a rate-limiting enzyme of triacylglycerol (TAG) biosynthesis in many organisms including algae. Many algae have multiple DGAT genes in their genomes. It is crucial to clarify substrate specificity and activity of different DGATs for understanding their biological roles. The current in vitro DGAT assays involve predominantly the use of radiolabeled substrates, either acyl-CoA or diacylglycerol (DAG). The availability of limited radiolabeled substrates and technical difficulties to conduct radiolabeled experiments have limited the use of these assays. Therefore, an assay without the involvement of radiolabeled substrates is needed. In the present study, we developed a novel in vitro DGAT assay using non-radiolabeled substrates and optimized its conditions including buffer pH and concentration, Mg2+ concentration, microsomal protein amount, acyl-CoA concentration, and incubation time. CrDGTT1, a type 2 DGAT from Chlamydomonas reinhardtii, was used to assess the feasibility of our non-radiolabeled in vitro assay toward different acyl-CoAs and DAGs. In addition, the substrate preference and activity of ScDGA1, a yeast-derived type 2 DGAT, were evaluated with our assay method, and the results obtained were consistent with those from a previous radiolabeled assay. We also demonstrated the suitability of this assay for the activity of phospholipid:diacylglycerol acyltransferase, an enzyme responsible for the acyl-CoA-independent TAG biosynthesis. Taken together, the in vitro acyltransferase assay developed here eliminates the use of radiolabeled substrates, is simple and reproducible, and allows the investigation of enzyme specificity and activity over a wide range of substrates.