A Single Glycosidase Harnesses Different Pyranoside Ring Transition State Conformations for Hydrolysis of Mannosides and Glucosides

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• 作者：Anupong Tankrathok ; Javier Iglesias-Fern谩ndez ; Rohan J. Williams ; Salila Pengthaisong ; Supaporn Baiya ; Zalihe Hakki ; Robert C. Robinson ; Maria Hrmova ; Carme Rovira ; Spencer J. Williams ; James R. Ketudat Cairns
• 刊名：ACS Catalysis
• 出版年：2015
• 出版时间：October 2, 2015
• 年：2015
• 卷：5
• 期：10
• 页码：6041-6051
• 全文大小：683K
• ISSN：2155-5435

文摘

Hydrolysis of 尾-d-mannosides by 尾-mannosidases typically proceeds via a B_2,5 transition state conformation for the pyranoside ring, while that of 尾-d-glucosides by 尾-glucosidases proceeds through a distinct ⁴H₃ transition state conformation. However, rice Os7BGlu26 尾-glycosidase hydrolyzes 4-nitrophenyl 尾-d-glucoside and 尾-d-mannoside with similar efficiencies. The origin of this dual substrate specificity was investigated by kinetic, structural, and computational approaches. The glycosidase inhibitors glucoimidazole and mannoimidazole inhibited Os7BGlu26 with K_i values of 2.7 nM and 10.4 渭M, respectively. In X-ray crystal structures of complexes with Os7BGlu26, glucoimidazole bound to the active site in a ⁴E conformation, while mannoimidazole bound in a B_2,5 conformation, suggesting different transition state conformations. Moreover, calculation of quantum mechanics/molecular mechanics (QM/MM) free energy landscapes showed that 4-nitrophenyl 尾-d-glucoside adopts a ¹S₃/⁴E conformation in the Michaelis complex, while 4-nitrophenyl 尾-d-mannoside adopts a ¹S₅/B_2,5 conformation. The QM/MM simulations of Os7BGlu26 catalysis of hydrolysis also supported the itineraries of ¹S₃ 鈫?⁴E/⁴H₃^{猝?/sup> 鈫?4C₁ for 尾-d-glucosides and 1S₅ 鈫?B_2,5猝?/sup> 鈫?OS₂ for 尾-d-mannosides, thereby revealing that a single glycoside hydrolase can hydrolyze glycosides of different configurations via distinct transition state pyranoside conformations.}