Pengthaisong Salila, Piniello Beatriz, Davies Gideon J, Rovira Carme, Ketudat Cairns James R
School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
ACS Catal. 2023 Apr 14;13(9):5850-5863. doi: 10.1021/acscatal.3c00620. eCollection 2023 May 5.
Retaining glycoside hydrolases use acid/base catalysis with an enzymatic acid/base protonating the glycosidic bond oxygen to facilitate leaving-group departure alongside attack by a catalytic nucleophile to form a covalent intermediate. Generally, this acid/base protonates the oxygen laterally with respect to the sugar ring, which places the catalytic acid/base and nucleophile carboxylates within about 4.5-6.5 Å of each other. However, in glycoside hydrolase (GH) family 116, including disease-related human acid β-glucosidase 2 (GBA2), the distance between the catalytic acid/base and the nucleophile is around 8 Å (PDB: 5BVU) and the catalytic acid/base appears to be above the plane of the pyranose ring, rather than being lateral to that plane, which could have catalytic consequences. However, no structure of an enzyme-substrate complex is available for this GH family. Here, we report the structures of β-glucosidase (GH116) D593N acid/base mutant in complexes with cellobiose and laminaribiose and its catalytic mechanism. We confirm that the amide hydrogen bonding to the glycosidic oxygen is in a perpendicular rather than lateral orientation. Quantum mechanics/molecular mechanics (QM/MM) simulations of the glycosylation half-reaction in wild-type GH116 indicate that the substrate binds with the nonreducing glucose residue in an unusual relaxed chair at the subsite. Nevertheless, the reaction can still proceed through a half-chair transition state, as in classical retaining β-glucosidases, as the catalytic acid D593 protonates the perpendicular electron pair. The glucose C6OH is locked in a , orientation with respect to the C5-O5 and C4-C5 bonds to facilitate perpendicular protonation. These data imply a unique protonation trajectory in Clan-O glycoside hydrolases, which has strong implications for the design of inhibitors specific to either lateral protonators, such as human GBA1, or perpendicular protonators, such as human GBA2.
保留型糖苷水解酶利用酸碱催化，酶的酸碱基团使糖苷键氧原子质子化，以促进离去基团离去，同时催化亲核试剂进攻形成共价中间体。一般来说，这种酸碱基团相对于糖环横向使氧原子质子化，使得催化酸碱基团和亲核羧酸根之间的距离在约4.5 - 6.5 Å 。然而，在糖苷水解酶（GH）家族116中，包括与疾病相关的人类酸性β-葡萄糖苷酶2（GBA2），催化酸碱基团和亲核试剂之间的距离约为8 Å（蛋白质数据银行：5BVU），并且催化酸碱基团似乎位于吡喃糖环平面上方，而不是该平面的横向，这可能会产生催化影响。然而，该GH家族没有酶 - 底物复合物的结构。在这里，我们报告了β-葡萄糖苷酶（GH116）D593N酸碱突变体与纤维二糖和层二糖形成复合物的结构及其催化机制。我们证实与糖苷氧原子形成的酰胺氢键呈垂直而非横向取向。野生型GH116糖基化半反应的量子力学/分子力学（QM/MM）模拟表明，底物在亚位点以异常松弛的椅式构象与非还原葡萄糖残基结合。尽管如此，反应仍可通过半椅式过渡态进行，如同经典的保留型β-葡萄糖苷酶一样，因为催化酸D593使垂直电子对质子化。葡萄糖C6OH相对于C5 - O5和C4 - C5键锁定在特定取向，以促进垂直质子化。这些数据暗示了O族糖苷水解酶中独特的质子化轨迹，这对于设计针对横向质子化酶（如人类GBA1）或垂直质子化酶（如人类GBA2）的特异性抑制剂具有重要意义。
