Ricinus communis contains an acyl-CoA synthetase that preferentially activates ricinoleate to its CoA thioester

As part of our effort to identify enzymes that are critical for producing large amounts of ricinoleate in castor oil, we have isolated three cDNAs encoding acyl-CoA synthetase (ACS) in the castor plant. Analysis of the cDNA sequences reveals that two of them, designated RcACS 2 and RcACS 4, contain complete coding regions corresponding to 694 and 690 amino acids, respectively. The third cDNA, RcACS 1, encodes a truncated gene sequence. The RcACS 2 and RcACS 4 share 77% identity at the amino acid sequence level. Complementation tests showed that both RcACS 2 and RcACS 4 successfully restored growth of a yeast mutant strain (YB525) deficient in ACS. Lysates from yeast cells expressing RcACS 2 and 4 were enzymatically active when using 14C-labeled oleic acid as a substrate. A cell fractionation study indicates that RcACS 2 and 4 are mainly associated with membranes. Substrate specificity assays indicate that the RcACS 2 preferentially activates ricinoleate, while the RcACS 4 has a preference for nonhydroxy fatty acids.