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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >MODIFICATION OF THE SUBSTRATE SPECIFICITY OF AN ACYL-ACYL CARRIER PROTEIN THIOESTERASE BY PROTEIN ENGINEERING
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MODIFICATION OF THE SUBSTRATE SPECIFICITY OF AN ACYL-ACYL CARRIER PROTEIN THIOESTERASE BY PROTEIN ENGINEERING

机译：蛋白质工程对酰基-酰基载体蛋白质硫酯酶的底物特异性的修饰

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The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coil and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated, We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1, This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression, We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other, Our results show that the C-terminal two-thirds of the protein is critical for the specificity, By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231R, by itself does not effect the specificity, However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0, Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of K-m,K-app with respect to 14:0-ACP, Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired, The ability to modify TE specificity should allow the production of additional ''designer oils'' in genetically engineered plants. [References: 31]
机译：植物酰基-酰基载体蛋白（ACP）硫酯酶（TEs）具有生物化学意义，因为它们在脂肪酸合成中的作用及其在植物种子油的生物工程中的用途。当来自加利福尼亚湾的编码12：0-ACP TE（Uc FatB1）的FatB1 cDNA在拟南芥和拟南芥和甘蓝型油菜的油菜种子中表达时，Umbellularia californica（Uc）表达了大量的月桂酸酯（12： 0）和少量的肉豆蔻酸盐（14：0）积累，我们从樟脑（Cinnamomum camphorum）（Cc）种子中分离了TE cDNA，该种子与Uc FatB1具有92％的氨基酸同一性，该TE，Cc FatB1主要水解如大肠杆菌表达所显示的14：0-ACP，我们已经研究了N和C末端区域在通过构建两种嵌合酶（其中一种蛋白质的N末端部分与之融合）来确定底物特异性中的作用。我们的结果表明，蛋白质的C末端的三分之二对于特异性至关重要。通过定点诱变，我们已使用Cc FatB1序列替换了Uc FatB1中的几个氨基酸作为指导。一个双突变体，将Met-197变为Arg，将Arg-199变为His（M197R / R199H），将Uc FatB1变成12：0/14：0 TE，对两种底物均具有相同的优先级。另一个突变T231R本身不影响特异性，但是，当与双重突变体结合以产生三重突变体（M197R / R199H / T231K）时，Uc FatB1会转化为14：0-ACP TE。双突变cDNA在缺乏脂肪酸降解作用的大肠杆菌K27中的表达导致相似量的12：0和14：0积累，而表达三突变cDNA的大肠杆菌主要产生14 ：0：非常少量的12：0。动力学研究表明，野生型Uc FatB1和三重突变体相对于14：0-ACP具有相似的Km，K-app值，抑制性研究也表明12：0-ACP是相对于14：0-ACP的良好竞争性抑制剂野生型和三重突变体均具有14：0-ACP。这些结果表明12：0-和14：0-ACP都可以同等地很好地结合两种蛋白质，但是在三重突变体的情况下，12：0-ACP的水解会严重受损。特异性应允许在基因工程植物中生产其他“设计油”。 [参考：31]

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