ABSTRACT
Fatty acids are activated in an ATP-dependent manner before they are utilized. We describe here how the 10 S triacylglycerol biosynthetic multienzyme complex from Rhodotorula glutinis is capable of activating non-esterified fatty acids for the synthesis of triacylglycerol. The photolabelling of the complex with [(32)P]azido-ATP showed labelling of a 35 kDa polypeptide. The labelled polypeptide was identified as acyl-acyl carrier protein (ACP) synthetase, which catalyses the ATP-dependent ligation of fatty acid with ACP to form acyl-ACP. The enzyme was purified by successive PAGE separations to apparent homogeneity from the soluble fraction of oleaginous yeast and its apparent molecular mass was 35 kDa under denaturing and reducing conditions. Acyl-ACP synthetase was specific for ATP. The K(m) values for palmitic, stearic, oleic and linoleic acids were found to be 42.9, 30.4, 25.1 and 22.7 microM, respectively. The antibodies to acyl-ACP synthetase cross-reacted with Escherichia coli acyl-ACP synthetase. Anti-ACP antibodies showed no cross-reactivity with the purified acyl-ACP synthetase, indicating no bound ACP with the enzyme. Immunoprecipitations with antibodies to acyl-ACP synthetase revealed that this enzyme is a part of the 10 S triacylglycerol biosynthetic complex. These results demonstrate that the soluble acyl-ACP synthetase plays a novel role in activating fatty acids for triacylglycerol biosynthesis in oleaginous yeast.
Subject(s)
Acyl Carrier Protein/metabolism , Carbon-Sulfur Ligases/chemistry , Carbon-Sulfur Ligases/isolation & purification , Rhodotorula/enzymology , Triglycerides/biosynthesis , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Carbon-Sulfur Ligases/metabolism , Cytidine Triphosphate/metabolism , Enzyme Activation , Fatty Acids/metabolism , Guanosine Triphosphate/metabolism , Kinetics , Molecular Sequence Data , Multienzyme Complexes/chemistry , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Uridine Triphosphate/metabolismABSTRACT
Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.
