As the major and abundant type of glucosinolates (GL) in plants, sinigrin has potential functions in promoting health and insect defense. The final step in the biosynthesis of sinigrin core structure is highly representative in GL compounds, which corresponds to the process from 3-methylthiopropyl ds-GL to 3-methylthiopropyl GL catalyzed by sulfotransferase (SOT). However, due to the lack of the crystallographic structure of SOT complexed with the 3-methylthiopropyl GL, little is known about this sulfonation process. Fortunately, the crystal structure of SOT 18 from Arabidopsis thaliana (AtSOT18) containing the substance (sinigrin) similar to 3-methylthiopropyl GL has been determined. To understand the enzymatic mechanism, we employed molecular dynamics (MD) simulation and quantum mechanics combined with molecular mechanics (QM/MM) methods to study the conversion from ds-sinigrin to sinigrin catalyzed by AtSOT18. The calculated results demonstrate that the reaction occurs through a concerted dissociative mechanism. Moreover, Lys93, Thr96, Thr97, Tyr130, His155, and two enzyme peptide chains (Pro92-Lys93 and Gln95-Thr96-Thr97) play a role in positioning the substrates and promoting the catalytic reaction by stabilizing the transition state geometry. Particularly, His155 acts as a catalytic base while Lys93 acts as a catalytic acid in the reaction process. The presently proposed concerted dissociative mechanism explains the role of AtSOT18 in sinigrin biosynthesis, and could be instructive for the study of GL biosynthesis catalyzed by other SOTs.

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芥子苷生物合成酶促机制的QM/MM研究

作为植物中主要和丰富的硫代葡萄糖苷（GL）类型，黑芥子苷具有促进健康和防虫的潜在功能。黑芥子核心结构生物合成的最后一步在 GL 化合物中具有高度代表性，这对应于磺基转移酶 (SOT) 催化的 3-甲基硫丙基 ds-GL 到 3-甲基硫丙基 GL 的过程。然而，由于缺乏与 3-甲硫丙基 GL 复合的 SOT 的晶体结构，因此对该磺化过程知之甚少。幸运的是，来自拟南芥的 SOT 18 的晶体结构(AtSOT18) 含有类似于 3-甲硫基丙基 GL 的物质 (sinigrin)。为了了解酶促机制，我们采用分子动力学 (MD) 模拟和量子力学结合分子力学 (QM/MM) 方法来研究 AtSOT18 催化的 ds-sinigrin 向sinigrin 的转化。计算结果表明，反应是通过协同的解离机制发生的。此外，Lys93、Thr96、Thr97、Tyr130、His155 和两条酶肽链（Pro92-Lys93 和 Gln95-Thr96-Thr97）通过稳定过渡态几何结构在定位底物和促进催化反应中发挥作用。特别是，His155 充当催化碱，而 Lys93 在反应过程中充当催化酸。