Molecular Mechanism of the Glycosylation Step Catalyzed by Golgi α-Mannosidase II: A QM/MM Metadynamics Investigation

Abstract

Golgi alpha-mannosidase II (GMII), a member of glycoside hydrolase family 38, cleaves two mannosyl residues from GlcNAcMan(5)GlcNAc(2) as part of the N-linked glycosylation pathway. To elucidate the molecular and electronic details of the reaction mechanism, in particular the conformation of the substrate at the transition state, we performed quantum mechanics/molecular mechanics metadynamics simulations of the glycosylation reaction catalyzed by GMII. The calculated free energy of activation for mannosyl glycosylation (23 kcal/mol) agrees very well with experiments, as does the conformation of the glycon mannosyl ring in the product of the glycosylation reaction (the covalent intermediate). In addition, we provide insight into the electronic aspects of the molecular mechanism that were not previously available. We show that the substrate adopts an (O)S(2)/B(2,5) conformation in the GMII Michaelis complex and that the nucleophilic attack occurs before complete departure of the leaving group, consistent with a D(N)A(N) reaction mechanism. The transition state has a clear oxacarbenium ion (OCI) character, with the glycosylation reaction following an (O)S(2)/B(2,5) --> B(2,5) [TS] --> (1)S(5) itinerary, agreeing with an earlier proposal based on comparing alpha- and beta-mannanases. The simulations also demonstrate that an active-site Zn ion helps to lengthen the O2'-H(O2') bond when the substrate acquires OCI character, relieving the electron deficiency of the OCI-like species. Our results can be used to explain the potency of recently formulated GMII anticancer inhibitors, and they are potentially relevant in deriving new inhibitors.

Mutational Analysis of Divalent Metal Ion Binding in the Active Site of Class II α-Mannosidase from Sulfolobus solfataricus

“…4 The efficiency of a divalent metal ion in activating ManA is probably a result of the ion's ability, via its coordination sphere, to stabilize the pyranose conformation of the −1 mannosyl and introduce the highenergy tension during transition state formation. 3,16 Compared to the wild-type enzyme, we do not observe dramatic changes for mutant enzymes H228E, H228Q, and H533Q in the K M for pNP-mannoside, except in the presence of Mn 2+ for H228E and H228Q or Cd 2+ for the H533Q enzyme (Table 1 and Figure 3A). The overall trend for these mutant enzymes seems to be that they are mainly affected in the binding of the metal ion as indicated by the increase in K A from 20-fold to more than several orders of magnitude (Table 2 and Figure 3B), when compared to that of the wild-type enzyme.…”

Glycosidases – A Mechanistic Overview

“…Furthermore, QM/MM metadynamics simulations also demonstrate that the zinc ion helps to lengthen the C2 hydroxyl bond when the substrate acquires the oxocarbenium character, facilitating the electron reduction of this species. 105 A similar role has been proposed for the calcium ion present in the structure of the endoplasmic reticulum α-mannosidase I from the GH47 family. The crystallographic structure shows that the cation coordinates with the hydroxyl groups that are attached to carbons C2 and C3 of 1-deoxymannojirimycin or kifunensin inhibitors.…”

“…Nevertheless, over the years, this diagram has been actively used as an "itinerary map" to design new enzyme inhibitors for therapeutic activities. In this regard, the conformational itinerary pathway of several GHs families has been studied, such as GH29 enzymes and α-xylosidases from the GH31 family that catalyze the hydrolysis using the 4 C1 ↔ 3 H4 ↔ 3 S1 glycosylation itinerary 93,103 ; inverting endoglucanases from the GH8 family that use the β-2 S0/ 2,5 B ↔ 2,5 B ↔ α-5 S1 glycosylation itinerary of the glycon ring 104 ; the glycosylation reaction of golgi α-mannosidase II from the GH38 family following an 0 S2/B2,5 ↔ B2,5 ↔ 1 S5 itinerary 105 ; the catalytic itinerary of 1,3-1,4-β-glucanase from the GH16 family 16 pursue the 1,4 B/ 1 S3 ↔ 4 E/ 4 H3 ↔ 4 C1 95,106 itinerary, and the inverting α-mannosidases from the GH47 family that follow the 3 S1 ↔ 3 H4 ↔ 4 C1 glycosylation itinerary. 107 Many GHs also contain cations in the region of the active site.…”

Tailoring recombinant protein quality by rational media design

“…In addition to kifunensine other inhibitors such as swainsonine and mannoimidazole inhibit mannosidases as well 98,99 Mannostatin A, a reversible and competitive inhibitor of Golgi α-mannosidase II, belongs to the most potent inhibitors of this enzyme 100,101 . Zinc plays an important catalytic role in the hydrolyzation step involving α-mannosidase II, as it helps to stabilize the transition state by relieving the electron deficiency of the Michaelis complex 102 . On the opposite, another study concluded that class II α-mannosidase is neither metal ion dependent nor inactivated by EDTA 94 .…”