Purification and characterization of acyl-acyl carrier protein synthetase from oleaginous yeast and its role in triacylglycerol biosynthesis.
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.
Gangar
A
A
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Karande
A A
AA
Rajasekharan
R
R
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019730:Carbon-Sulfur Ligases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003570:Cytidine Triphosphate / Q000378:metabolism; D004789:Enzyme Activation; D005227:Fatty Acids / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology*; D014280:Triglycerides / Q000096:biosynthesis*; D014544:Uridine Triphosphate / Q000378:metabolism
D000213:Acyl Carrier Protein; D005227:Fatty Acids; D009097:Multienzyme Complexes; D014280:Triglycerides; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase; D014544:Uridine Triphosphate