Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
ACS Chem Biol. 2022 Sep 16;17(9):2507-2518. doi: 10.1021/acschembio.2c00408. Epub 2022 Aug 29.
Toxins TcdA and TcdB from glucosylate human colon Rho GTPases. TcdA and TcdB glucosylation of RhoGTPases results in cytoskeletal changes, causing cell rounding and loss of intestinal integrity. Clostridial toxins TcdA and TcdB are proposed to catalyze glucosylation of Rho GTPases with retention of stereochemistry from UDP-glucose. We used kinetic isotope effects to analyze the mechanisms and transition-state structures of the glucohydrolase and glucosyltransferase activities of TcdB. TcdB catalyzes Rho GTPase glucosylation with retention of stereochemistry, while hydrolysis of UDP-glucose by TcdB causes inversion of stereochemistry. Kinetic analysis revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an S mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mechanical calculations revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an S mechanism with the formation of a distinct oxocarbenium phosphate ion pair transition state where the glycosidic bond to the UDP leaving group breaks prior to attack of the threonine nucleophile from Rho GTPase.
毒素 TcdA 和 TcdB 使人类结肠 Rho GTPases 发生葡糖基化。TcdA 和 TcdB 使 RhoGTPases 葡糖基化会导致细胞骨架发生变化，从而导致细胞变圆和肠道完整性丧失。产志贺毒素的梭菌毒素 TcdA 和 TcdB 被认为能催化 Rho GTPases 的葡糖基化，保留 UDP-葡萄糖的立体化学结构。我们使用动力学同位素效应分析了 TcdB 的糖基水解酶和葡糖基转移酶活性的机制和过渡态结构。TcdB 以保留立体化学的方式催化 Rho GTPase 的葡糖基化，而 TcdB 水解 UDP-葡萄糖会导致立体化学的反转。动力学分析表明 TcdB 通过形成三元复合物进行葡糖基化，没有中间产物，支持亲核进攻和离去基团离开发生在葡萄糖环同一面上的 S 机制。动力学同位素效应结合量子力学计算揭示了 TcdB 的糖基水解酶和葡糖基转移酶活性的过渡态都具有高度离解性。具体来说，TcdB 的葡糖基转移酶反应通过 S 机制进行，形成独特的氧碳正离子磷酸二酯键过渡态，其中 UDP 离去基团的糖苷键在 Rho GTPase 的苏氨酸亲核进攻之前断裂。
