Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an S_Ni Mechanism and Ion Pair Transition State

Abstract

Toxins TcdA and TcdB from Clostridioides difficile 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 N i 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 N i 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.

Catalytic Mechanism of Nonglycosidic C–N Bond Formation by the Pseudoglycosyltransferase Enzyme VldE

“…To determine whether the coupling reaction by VldE takes place via an S N i -like mechanism or a 1,4-elimination–1,4-addition mechanism, we performed kinetic isotope effect (KIE) studies using GDP-valienol, GDP-[1- 2 H]­valienol, GDP-[2- 2 H]­valienol, and GDP-[7- 2 H 2 ]­valienol. KIE has been used to determine the catalytic mechanism of several GTs, − including OtsA, which catalyzes the formation of trehalose 6-phosphate through an S N i -like mechanism . Similar to the case for OtsA, the VldE-catalyzed reaction is practically irreversible under the assay conditions, eliminating the possibility of a reverse reaction as a commitment factor.…”

Isofagomine inhibits multiple TcdB variants and protects mice fromClostridioides difficileinduced mortality

“…Here, we test whether isofagomine exhibits similar inhibitory activity against other TcdB variants. TcdB-GTD variants 1-8 were expressed and purified and kinetic constants for substrates UDP-glucose and Rac1 were determined using a radiolabel incorporation assay which measures the amount of glycosylated Rac1 24,25 . The Michaelis-Menten ( K m ) constant for UDP-glucose was similar for each variant and ranged from 4.3 μM to 9.4 μM (Table 1).…”

“…The efficacy of isofagomine was tested against major and multiple variants of TcdB (TcdB variants 1-8). Isofagomine inhibits the GTD of TcdA and TcdB by mimicking the oxocarbenium ion transition state formed during the TcdA and TcdB GTD reaction 24,25 . Isofagomine inhibited the GTD activity of all TcdB variants tested.…”

“…TcdB-GTD variants were expressed in Escherichia coli (One Shot BL21 Star (DE3)) cell line. Conditions for protein expression and protein purification of TcdB1-8-GTD were carried out as described previously 24,25 with purification by Ni-NTA chromatography and protein preparation in 20 mM Tris pH 7.5, 150 mM NaCl, 15% v/v glycerol, freezing and storage at -80 °C.…”

“…Previously, we reported isofagomine as an inhibitor of the GTD’s of TcdA1 and TcdB1 24 . Isofagomine is a transition state analogue that mimics the positively charged glucocation formed at the transition state of the TcdA and TcdB glycosyltransferase reactions 25 . Isofagomine binds in the TcdA and TcdB active sites and forms an ion-pair interaction with the negatively charged β-phosphate of UDP, a by-product of the Tcd glycosyltransferase reaction (Figure 2) 24,25 .…”