Computationally Guided Enzymatic Studies on Schizochytrium- Sourced Malonyl-CoA:ACP Transacylase
Computationally Guided Enzymatic Studies on Schizochytrium- Sourced Malonyl-CoA:ACP Transacylase
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DOI：
10.1021/acs.jafc.2c05447
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发表时间：
2022-10-20
影响因子：
6.1
通讯作者：
He, Ning
中科院分区：
文献类型：
--
作者：
Chi, Guoxiang;Cao, Xingyu;He, Ning
丙二酰-CoA：ACP转酰酶（MAT）结构域负责在真核微藻（裂殖壶菌属）和海洋细菌（Moritella marina、发光杆菌属（Photobacterium profundum）和希瓦氏菌属（Shewanella））中多不饱和脂肪酸（PUFA）的生物合成中选择和掺入丙二酰结构单元。阐明MAT的底物特异性和催化机理的结构基础将有助于提高PUFAs的产量和质量。在这里，分子动力学模拟指导的方法进行了鉴定和突变的特异性赋予裂殖壶菌MAT域内的残基。结合诱变、无细胞蛋白质合成和体外生物化学测定，我们剖析了与结合和催化相关的附近相互作用和分子机制，发现Ser 154 C β-O γ键的重定向建立了用于催化的独特质子转移链（His 153-Ser 154和Asn 235-His 153-Ser 154）。Gln 66可以被酪氨酸取代，以缩短His 153（N-2）和Ser 154（O-γ）之间的距离，这有助于更快的质子转移速率，允许比野生型更好地利用酰基底物。此外，我们筛选了一种突变体，显示PUFA积累增加18.4%。这些发现为通过蛋白质工程研究MAT提供了重要的见解，并将有利于解剖其他PUFA相关催化结构域的分子机制。
The malonyl-CoA:ACP transacylase (MAT) domain is responsible for the selection and incorporation of malonyl building blocks in the biosynthesis of polyunsaturated fatty acids (PUFAs) in eukaryotic microalgae (Schizochytrium) and marine bacteria (Moritella marina, Photobacterium profundum, and Shewanella). Elucidation of the structural basis underlying the substrate specificity and catalytic mechanism of the MAT will help to improve the yield and quality of PUFAs. Here, a methodology guided by molecular dynamics simulations was carried out to identify and mutate specificity-conferring residues within the MAT domain of Schizochytrium. Combining mutagenesis, cell-free protein synthesis, and in vitro biochemical assay, we dissected nearby interactions and molecular mechanisms relevant for binding and catalysis and found that the reorientation of the Ser154 C beta-O gamma bond establishes distinctive proton-transfer chains (His153-Ser154 and Asn235-His153-Ser154) for catalysis. Gln66 can be replaced by tyrosine to shorten the distance between His153 (N epsilon 2) and Ser154 (O gamma), which facilitates a faster proton-transfer rate, allowing better use of acyl substrates than the wild type. Furthermore, we screened a mutant that displayed an 18.4% increase in PUFA accumulation. These findings provide important insights into the study of MAT through protein engineering and will benefit dissecting the molecular mechanisms of other PUFA-related catalytic domains.