Structural Insight into Acyl-ACP Thioesterase toward Substrate Specificity Design
Structural Insight into Acyl-ACP Thioesterase toward Substrate Specificity Design
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酰基 ACP 硫酯酶的结构洞察以实现底物特异性设计
DOI：
10.1021/acschembio.7b00641
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发表时间：
2017-11-01
影响因子：
4
通讯作者：
Xue, Song
中科院分区：
文献类型：
--
作者：
Feng, Yanbin;Wang, Yayue;Xue, Song
酰基-ACP硫酯酶（TE）在II型脂肪酸合成期间催化硫酯键的水解并直接决定脂肪酸链长度。大多数TE负责识别16：0和18：1底物，而特定的TE中断C8 C14处的酰基-ACP延伸。然而，迄今为止，TE的酰基选择机制尚未得到彻底阐明。在这项研究中，C12-特异性硫酯酶FatB从加州伞形，它由两个独立的热狗域，晶体结构进行了测定。一个不规范的Asp-His-Glu催化网络被确定为C-末端热狗结构域，而底物结合口袋被确定为N-末端热狗结构域。此外，我们阐明了UcFatBs底物选择机制，该机制由β 5，β 2和β 4折叠上的几个非保守氨基酸调节，并由α 1螺旋上的T137包围。在此基础上，通过调节底物结合口袋容量，对C12特异性TE进行了合理的重新设计，使其具有C14选择性。T137 G突变体在体外表现出与C12底物相比对C14底物的相对活性。此外，重组UcFatB_T137G在工程化的E.大肠杆菌细胞。TE的底物选择机制的揭示为脂肪酸合成提供了一种新的策略。
Acyl-ACP thioesterase (TE) catalyzes the hydrolysis of thioester bonds during type II fatty acid synthesis and directly determines fatty acid chain length. Most TEs are responsible for recognition of 16:0 and 18:1 substrates, while specific TEs interrupt acyl-ACP elongation at C8C14. However, the acyl selection mechanism of TE has not been thoroughly elucidated to date. In this study, the crystal structure of the C12-specific thioesterase FatB from Umbellularia californica, which consists of two independent hotdog domains, was determined. An uncanonical Asp-His-Glu catalytic network was identified on the C-terminal hotdog domain, whereas the substrate binding pocket was determined to be on the N-terminal hotdog domain. Moreover, we elucidated UcFatBs substrate selection mechanism, which is accommodated by several unconservative amino acids on the beta 5, beta 2, and beta 4 sheets and enclosed by T137 on the alpha 1 helix. On this basis, the C12-specific TE was rationally redesigned toward C14 selectivity by tuning the substrate binding pocket capacity. The T137G mutant demonstrated comparative relative activity on C14 substrates compared to C12 substrates in vitro. Furthermore, the reconstructed UcFatB_T137G achieved C14 fatty acid content up to 40% in contrast to 10% C14 from the wild type in engineered E. coli cells. The unraveled substrate selection mechanism of TE provides a new strategy for tailoring fatty acid synthesis.