RESUMO
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.
