Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas
Structural and functional analyses of a saturated acyl ACP thioesterase, type B from immature seed tissue of Jatropha curcas
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DOI：
10.1111/j.1438-8677.2010.00410.x
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发表时间：
2011-05-01
期刊：
影响因子：
3.9
通讯作者：
Kush, A.
中科院分区：
文献类型：
--
作者：
Dani, K. G. Srikanta;Hatti, K. S.;Kush, A.
植物酰基-酰基载体蛋白（ACP）硫酯酶的独特结构和功能域及其与ACP连接的脂肪酸底物复合物的复杂相互作用仍然是难以捉摸的。E.迄今为止报道的许多植物硫酯酶的基于大肠杆菌的异源表达和表征尚未被扩展并与计算机模拟研究相联系，以解释植物硫酯酶底物特异性的多样性。本研究从小桐子未成熟种子组织中分离到一种硫酯酶cDNA，该硫酯酶cDNA为B型，在大肠杆菌中表达时对硬脂酰酰基ACP具有特异性。coliK 27 fadD 88，脂质利用突变株。分离的JcFatB蛋白的选定区域的同源性建模和分子对接预测，它对硬脂酸酯（18：0）和棕榈酸酯（16：0）具有高亲和力。序列的结构分析证实了在多个步骤中加工的转运肽的存在。该酶定位于叶绿体中，并具有B型植物硫酯酶特征的N-末端内叶绿体跨膜结构域。配体与JcFatB的对接及其与模拟的麻风树硫酯酶A型的比较为麻风树硫酯酶的天然底物偏好提供了进一步的证据。这项研究提供了必要的线索，开发未来的方法，大规模的细菌生产的游离脂肪酸和设计的策略，以调节在这个重要的非食用，种子油植物的种子油组成。
The distinguishing structural and functional domains of plant acyl-acyl carrier protein (ACP) thioesterases and their complex interaction with the ACP-linked fatty acid substrate complex have remained elusive. E. coli based heterologous expression and characterisation of many plant thioesterases reported so far have not been extended and linked to in silico modelling studies to explain the diversity in plant thioesterase substrate specificities. In this study, a thioesterase cDNA isolated from immature seed tissues of Jatropha curcas was found to be type B and specific to stearoyl acyl ACP when expressed in E. coli K27fadD88, a lipid utilisation mutant. Homology modelling and molecular docking of a selected region of the isolated JcFatB protein predicted that it had high affinity towards both stearate (18:0) and palmitate (16:0). Structural analysis of the sequence confirmed the presence of a transit peptide that is processed in multiple steps. The enzyme is localised in the chloroplasts and has an N-terminal inner chloroplast transmembrane domain characteristic of type B plant thioesterases. Docking of ligands with JcFatB and its comparison with a modelled Jatropha thioesterase type A provided further evidence for native substrate preferences of Jatropha thioesterases. This study provides essential clues to develop future methods for large-scale bacterial production of free fatty acids and for design of strategies to modulate the seed oil composition in this important non-edible, seed oil plant.