Separation and Characterization of Two Long‐Chain Acyl‐CoA Synthetases from <i>Candida lipolytica</i>

Abstract

Two activities of long‐chain acyl‐CoA synthetase have been demonstrated in the particulate fraction derived from cells of Candida lipolytica . One is observed both in oleic‐acid‐grown and in glucose‐grown cells and is independent of phosphatidylcholine. The other is found only in oleicacid‐grown cells and is dependent on phosphatidylcholine. The phosphatidylcholine‐independent activity is more resistant to heat or Triton X‐100 than the phosphatidylcholine‐dependent activity. Moreover, the phosphatidylcholine‐independent activity is readily resolved from the particulate fraction by Triton X‐100 treatment, whereas the phosphatidylcholine‐dependent activity remains in the particulate fraction after this treatment. Thus, the two activities have been separated from each other. These results indicate that C. lipolytica cells grown on·oleic acid possess two distinct long‐chain acyl‐CoA synthetases. The phosphatidylcholine‐independent enzyme is designated as acyl‐CoA synthetase I, and the phosphatidylcholine‐dependent enzyme as acyl‐CoA synthetase II. The presence of two distinct long‐chain acyl‐CoA synthetases is further supported by the occurrence of mutant strains of C. lipolytica that are defective in acyl‐CoA synthetase I but possess acyl‐CoA synthetase II. Acyl‐CoA synthetase II exhibits a broader substrate specificity with respect to fatty acid than acyl‐CoA synthetase I. This, together with the inducible nature of acyl‐CoA synthetase II, supports the concept, based on the phenotype of the mutants mentioned above, that acyl‐CoA synthetase I is responsible for the production of acyl‐CoA to be utilized for the synthesis of cellular lipids, while acyl‐CoA synthetase II provides acyl‐CoA which is degraded to yield acetyl‐CoA [Kamiryo, T., Mishina, M., Tashiro, S. &amp; Numa, S. (1977) Proc. Natl Acad. Sci. U.S.A. 74 , 4947–4950].