QM/MM Study on Enzymatic Mechanism in Sinigrin Biosynthesis

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.     

Globo H四糖衍生物的高效合成

设计合成了2个Globo H四糖衍生物1和2, 将其作为标准样品可用于研究β1,3-葡萄糖醛酸(GlcA)转移酶及GlcA-3-O-硫酸化(Sulfo)转移酶在肿瘤组织内的特异性表达.     

Immobilizing sulfotransferase 1A1 on magnetic microparticles and their evaluation using capillary electrophoresis

Sulfotransferases are categorized as phase II metabolic enzymes. Human sulfotransferase 1A1 (SULT1A1) is involved in the sulfonation of xenobiotics with aid from the cofactor 3'‐phosphoadenosine‐5'‐phosphosulfate that acts as a sulfonate donor. In this study, we have attempted to immobilize SULT1A1 on magnetic microparticles (MMs). Different functionalized MMs were used to immobilize SULT1A1 and their enzyme activity was compared to the control (enzyme in solution). Paracetamol was used as model substrate. Separation of paracetamol and paracetamol sulfate by CE‐UV was optimized and validated. MMs with epoxy based immobilization of SULT1A1 showed better enzyme activity. Hence, they were tested for repeated usage to allow their implementation for the development of a CE immobilized micro enzyme reactor.     

Identification of 3-enol sulfate of Cypridina luciferin,Cypridina luciferyl sulfate,in the sea-firefly Cypridina (Vargula) hilgendorfii

Cypridina luciferin from the luminous ostracod Cypridina (Vargula) hilgendorfii has an imidazopyrazinone core structure (3,7-dihydroimidazopyrazin-3-one), which is identical to that of coelenterazine. Cypridina luciferyl sulfate (3-enol sulfate of Cypridina luciferin) was isolated for the first time and the chemical structure was identified by LC/ESI–TOF–MS analysis. Furthermore, Cypridina luciferyl sulfate was chemically synthesized, and its absorption and MS/MS spectra were in agreement with that of Cypridina luciferyl sulfate isolated. Using the crude extracts of Cypridina specimens, Cypridina luciferyl sulfate could be converted to Cypridina luciferin in the presence of adenosine 3′,5′-diphosphate (PAP), and Cypridina luciferin was converted to Cypridina luciferyl sulfate in the presence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS). These results suggested that a sulfotransferase catalyzes the reversible sulfation of Cypridina luciferin in Cypridina hilgendorfii. In aqueous solution, Cypridina luciferyl sulfate was more stable than Cypridina luciferin and might be a storage form of Cypridina luciferin.     

黑芥子苷生物合成中酶催化机理的QM/MM研究

黑芥子苷作为植物中硫代葡萄糖苷最为主要且丰富的类型,在促进健康和昆虫防御方面具有潜在的功能. 黑芥子苷核心结构生物合成的最后一步在硫代葡萄糖苷化合物中具有高度的代表性. 这一步是在硫转移酶的催化作用下,从3-(甲基硫代)丙基脱硫葡萄糖苷转化为3-(甲基硫代)丙基葡萄糖苷的过程. 由于硫转移酶与3-(甲基硫代)丙基葡萄糖苷复合物的晶体结构还未见报道,因此一直以来人们对该磺化反应的细节知之甚少. 幸运的是,黑芥子苷与3-(甲基硫代)丙基葡萄糖苷的结构十分相似,而且结合有黑芥子苷的拟南芥硫转移酶18的晶体结构已经确定. 为了了解此酶的作用机理,本文采用分子动力学模拟以及组合的量子力学和分子力学方法研究了拟南芥硫转移酶18催化脱硫-黑芥子苷转化为黑芥子苷的过程. 计算结果表明,该反应是通过协同解离机理发生的,而且在反应过程中,赖氨酸93、苏氨酸96、苏氨酸97、酪氨酸130、组氨酸155和酶的两条肽链(脯氨酸92-赖氨酸93肽链和谷氨酰胺95-苏氨酸96-苏氨酸97肽链)稳定了过渡态的结构,在定位底物和促进催化反应中发挥了重要的作用,其中,组氨酸155在反应过程中充当催化碱,赖氨酸93充当催化酸. 目前提出的协同解离机理解释了拟南芥硫转移酶18在黑芥子苷生物合成中的作用,并对其他硫转移酶催化葡萄糖苷生物合成的研究具有指导意义.