He Xiaohua, Chen Grace Q, Kang Sung T, McKeon Thomas A
Western Regional Research Center, USDA, 800 Buchanan St, Albany, CA 94710, USA.
Lipids. 2007 Oct;42(10):931-8. doi: 10.1007/s11745-007-3090-0. Epub 2007 Aug 7.
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.
作为我们鉴定蓖麻油中大量产生蓖麻醇酸所需关键酶的工作的一部分，我们从蓖麻植物中分离出了三个编码酰基辅酶A合成酶（ACS）的cDNA。对cDNA序列的分析表明，其中两个分别命名为RcACS 2和RcACS 4，包含完整的编码区，分别对应694和690个氨基酸。第三个cDNA，RcACS 1，编码一个截短的基因序列。RcACS 2和RcACS 4在氨基酸序列水平上具有77%的同一性。互补试验表明，RcACS 2和RcACS 4都成功地恢复了缺乏ACS的酵母突变菌株（YB525）的生长。当使用14C标记的油酸作为底物时，表达RcACS 2和4的酵母细胞裂解物具有酶活性。细胞分级分离研究表明，RcACS 2和4主要与膜相关。底物特异性分析表明，RcACS 2优先激活蓖麻醇酸，而RcACS 4更倾向于非羟基脂肪酸。
