S47: Structure-function analysis of diacylglycerol acyltransferase sequences for metabolic engineering and drug discovery

Diacylglycerol acyltransferase families (DGATs) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. Over-expression of DGATs increases TAG in plants. Therefore, understanding the roles of DGATs will provide information for therapeutic intervention for obesity and related diseases and help to create transgenic plants with value-added properties. The objective of this study was to identify conserved sequence motifs and amino acid residues for better understanding of the structure-function relationship of these important enzymes. 133 DGAT sequences from 83 organisms including plants, animals, fungi and human are analyzed by phylogenetic analysis and multiple sequence alignment. This study identified conserved sequence motifs and amino acid residues in all DGATs and the two subfamilies. The importance of conserved residues has been demonstrated by site-directed and natural mutants. None of DGATs from any species was heterogeneous produced in a bacterial expression system because they are integral membrane proteins and difficult to express and purify. We recently developed a procedure for full-length recombinant DGAT expression in E. coli. The sequence analysis and developed expression method should facilitate studying the structure-function relationship of DGATs with the ultimate goal to identify critical amino acid residues for engineering superb enzymes in metabolic engineering and selecting enzyme inhibitors in therapeutic application for obesity and related diseases.