title: Use of limited proteolysis and mutagenesis to identify folding domains and sequence motifs critical for wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase activity.
abstract: Triacylglycerols and wax esters are synthesized as energy storage molecules by some proteobacteria and actinobacteria under stress. The enzyme responsible for neutral lipid accumulation is the bifunctional wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT). Structural modeling of WS/DGAT suggests that it can adopt an acyl-CoA-dependent acyltransferase fold with the N-terminal and C-terminal domains connected by a helical linker, an architecture demonstrated experimentally by limited proteolysis. Moreover, we found that both domains form an active complex when coexpressed as independent polypeptides. The structural prediction and sequence alignment of different WS/DGAT proteins indicated catalytically important motifs in the enzyme. Their role was probed by measuring the activities of a series of alanine scanning mutants. Our study underscores the structural understanding of this protein family and paves the way for their modification to improve the production of neutral lipids.
authors.affiliation: Departamento de Biología Molecular e Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-SODERCAN, Santander, Spain.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Villa
authors.forename: Matilde
authors.initials: M
authors.lastname: Cabezas
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Gabriel
authors.initials: G
authors.lastname: Moncalián
mesh_terms: D039903:Actinobacteria / Q000201:enzymology / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics* / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017510:Protein Folding; D020560:Proteobacteria / Q000201:enzymology / Q000378:metabolism; D059748:Proteolysis
chemical_list: D000214:Acyl Coenzyme A; D004075:Diglycerides; D000217:Acyltransferases; D051048:Diacylglycerol O-Acyltransferase; C079165:long-chain-alcohol O-fatty-acyltransferase