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Structure-Based Mechanism and Specificity of Human Galactosyltransferase β3GalT5
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  • Jennifer M. Lo
    Jennifer M. Lo
    Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
    Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan
    Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan
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  • Chih-Chuan Kung
    Chih-Chuan Kung
    Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
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  • Ting-Jen Rachel Cheng
    Ting-Jen Rachel Cheng
    Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
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  • Chi-Huey Wong*
    Chi-Huey Wong
    Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
    Department of Chemistry, Scripps Research, La Jolla, California 92037, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-9961-7865
  • Che Ma*
    Che Ma
    Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
    Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-4741-2307
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2025, 147, 13, 10875–10885
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https://doi.org/10.1021/jacs.4c11724
Published March 25, 2025

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Abstract

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Human β1,3-galactosyltransferase 5 (β3GalT5) is a key enzyme involved in the synthesis of glycans on glycoproteins and glycolipids that are associated with various important biological functions, especially tumor malignancy and cancer progression, and has been considered as a promising target for development of anticancer agents. In this study, we determined the X-ray structures of β3GalT5 in complex with the stable donor analogue UDP-2-fluorogalactose or the native donor substrate UDP-galactose (UDP-Gal) and several glycan acceptors at different reaction steps. Based on the structures obtained from our experiments, β3GalT5 catalyzes the transfer of galactose from UDP-Gal to a broad spectrum of glycan acceptors with an SN2-like mechanism; however, in the absence of a glycan acceptor, UDP-Gal is slowly converted to UDP and two other products, one is galactose through an SN2-like mechanism with water as an acceptor and the other is an oxocarbenium-like product, presumably through an SN1-like mechanisms. The structure, mechanism, and specificity of β3GalT5 presented in this study advance our understanding of enzymatic glycosylation and provide valuable insights for application to glycan synthesis and drug design targeting β3GalT5-associated cancer.

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Introduction

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Human beta-1,3-galactosyltransferase 5 (β3GalT5) is a member of the glycosyltransferase-31 family which is expressed in various tissues (1,2) and catalyzes the β-1,3-galactosylation of glycans on glycoproteins and glycolipids. β3GalT5 accepts glycans with terminal galactose (Gal), N-acetylglucosamine (GlcNAc), or N-acetylgalactosamine (GalNAc), leading to various important biological functions, including development, immune response, and cancer progression. (3−7) While other family members of β3GalT show different substrate specificity for N- and O-linked glycans, β3GalT5 prefers core 3 structures, globo- and Thr O-glycans, as well as type 1 Lewis glycans as substrates, (2,7,8) and the products generated are often associated with tumor malignancy and cancer progression, thus termed as tumor-associated carbohydrate antigens (TACAs).
It has been observed that high expression of β3GalT5 correlates with advanced cancer progression and poor clinical outcome in breast, (3,5,9) pancreatic, (10,11) liver, (4,7) ovarian, (12) gastric, (13) and nonsmall cell lung (14) cancers and that β3GalT5 promotes cancer cell proliferation, migration, and invasion by regulating the expression of cell adhesion molecules and extracellular matrix proteins. For example, β3GalT5 catalyzes the galactosylation of type 1 chain N-acetyllactosamine glycan (GlcNAc-β1,3-Gal-) (Figures 1B and S1) to form Lewis a (Lea), Lewis b (Leb), and sialyl Lewis a (sLea). Notably, sLea, also known as carbohydrate antigen 19-9 (CA19-9), serves as a useful tumor marker for the detection of early stage cancer and is frequently accumulated in the sera of patients with colonic, gastric, and pancreatic cancer. (10,15) CA19-9 was also found to accelerate cancer progression through the PI3K/Akt/mTOR pathway and selectin-mediated signaling. (10) In the biosynthesis of globo-series glycosphingolipids, β3GalT5 catalyzes the transfer of Gal from UDP-Gal to the terminal GalNAc residue of globotetraosyl ceramide (Gb4cer) (Figures 1A and S1) to generate Gb5 (also known as stage-specific embryonic antigen-3, SSEA-3). SSEA-3 can be further converted to SSEA-4 or Globo-H. Many cancers exhibit enhanced expression of SSEA-3, SSEA-4, and Globo-H, making these three cancer-associated globo-series glycosphingolipids (GSLs) promising clinical targets for immunotherapy. (9,16−19) A recent study showed that SSEA-3, SSEA-4, and Globo-H formed a complex with FAK/CAV1/Akt/RIP for signaling through the AKT-associated EGFR pathway and promoted cancer cell survival and metastasis. (16) Knockdown of β3GalT5 expression in breast cancer cells caused dissociation of RIP from the complex, triggering cancer cell apoptosis. (16) Additionally, silencing β3GalT5 reduces the migration and invasion ability through the regulated β-catenin/ZEB1 pathway during the epithelial-to-mesenchymal transition process. In vivo studies using patient-derived xenograft transplanted mice models demonstrate that β3GalT5 not only promotes tumor growth but also stimulates lymph node and lung metastasis. (5)

Figure 1

Figure 1. Overall structure of human β3GalT5. (A) Synthesis of SSEA-3 from Gb4 catalyzed by β3GalT5. (B) β3GalT5 catalyzed galactosylation in the synthesis of Core 3 O-glycans, globo-series glycosphingolipids (GSLs), and lacto-series GSLs. (C) Construct of gene for β3GalT5 expression. (D) Overall structure of β3GalT5 with substrates: composite image with β3GalT5:UDP2FGal superimposed on β3GalT5:Gb4 glycan. The color gradient from light to dark blue indicates the protein structure from N-terminal to C-terminal. The divalent ion Mn2+ is colored purple, UDP is brown, donor sugar galactose is gold, and acceptor Gb4 glycan is green.

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We also demonstrated that the globo-series glycans are exclusively expressed in 15 types of cancers and their stem cells (20) and knockdown of β3GalT5 led to cancer cell apoptosis with no effect on normal cells. (16) This work has led to the development of therapeutic cancer vaccines targeting Globo-H and the other globo-series glycans. (21) Taken together, β3GalT5 plays a pivotal role in the synthesis of TACAs and is considered as a promising target for development of anticancer agents. In this study, we determined the X-ray structure of the luminal domain of human β3GalT5 and elucidated its mechanism and specificity with the goal of providing valuable guidelines to facilitate the development of structure-based anticancer agents. We determined the structures of human β3GalT5 at various stages (a total of 8 structures) of the enzymatic reaction, including the dissociated galactose transition-like structures, and identified a broad-spectrum acceptor substrate, manifested by detailed structural rationales of each step for elucidation of the reaction mechanism and laying the groundwork for rational drug design and better understanding of glycobiology.

Results and Discussion

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Overall Structure of β3GalT5

β3GalT5 is a member of the glycosyltransferase-31 (GT31) family of carbohydrate-active enzymes (CAZy) (22) and exhibits limited overall sequence identity (25–29%) to other family members. This limited sequence homology has hindered the establishment of robust structure–function relationships. (8) To address this issue, we expressed and purified β3GalT5 and cocrystallized it in the presence of UDP-galactose (UDP-Gal) and different substrates. Our structural investigations began by solving the luminal domain structure of human β3GalT5 using sulfur single-wavelength anomalous dispersion phasing, achieving a resolution of 2.20 Å (Figure S2A–E). Subsequently, we determined additional β3GalT5 structures in complex with various substrates or products using molecular replacement with the sulfur derivative structure serving as the template. In total, we obtained eight structures, including β3GalT5 in complex with UDP-Gal, Gb4 glycan, GlcNac-β1,3-Gal, GlcNAc-β1,3-GalNAc, and Man-β1,6-Man. All structures belong to the P1 21 1 space group, with each asymmetric unit containing two protein molecules (Figure S2F).
β3GalT5 contains 310 amino acids, including the cytosolic N-terminal sequence (residues 1–7), the transmembrane domain (residues 8–28), and the soluble catalytic domain (residues 29–310). The recombinant protein used for crystallization contains residues Phe31-Pro308 (Figure 1C), but only residues Asp41-Pro308 are well-defined in the electron density maps. The catalytic domain adopts a mixed α/β Rossmann-like fold commonly observed in the GT-A glycosyltransferases superfamily and consists of a seven-stranded β sheet core (β1, β2, β3, β4, β5, β6, and β7) surrounded primarily by α helices (α1, α2, α3, α4, α5, α6, and α7), a two-strand antiparallel β sheet (β5′ and β7’), (23) and an additional 13-residue α helix (α8) at the C terminus (Figure 1D). Notably, two disulfide bonds (Cys52-Cys146 and Cys276-Cys307) stabilize the structure, and of the three glycosylation sites (Asn130, Asn174, and Asn231), the Asn174 glycosite reveals a complete paucimannose structure with one core fucose, which stabilizes the crystal packing by anchoring between adjacent molecules (Figure S2G).
Regarding the structural homology of β3GalT5 to other described structures, the DALI server (24) reveals structural similarities with other galactosyltransferases, including β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2; e.g., PDB entries 6WMO, (25) 7JHN, (26) 8TJC (27)), D. melanogaster core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferse 1 (DmC1GalT1; PDB entries 7Q4I (28)), mouse manic fringe (Mfng; PDB entries 2J0A and 2J0B (29)), and A. fumigatus galactofuranosylransferase (AfGfsA; PDB entries 8YRL), all of which belong to the CAZy31 family. Despite their diverging sequence identity to β3GalT5 (29, 19, 17, and 13%, respectively) and differing acceptor substrates, the server yields favorable scores, suggesting good structural superimposition (rmsd of ∼1.7, ∼2.1, ∼2.3, and ∼3.0 Å, respectively; with superimposed residues ranging from 112 to 203 residues). For retaining GT, α-1,3-galactosyltransferase (α3GalT; PDB entries 5NRD (30)) also has comparable key interacting residues to those of inverting GTs. The rmsd value is around 3.9 Å when compared to β3GalT5 with 90 Cα-atoms. The common active site architecture can be observed among these structures, despite differences in their substrate binding and reaction mechanisms (Figure S3).

Substrates Binding Clefts

Both donor and acceptor sugars are located close to the center of the whole binding cleft (Figure 2A). During the cocrystallization of UDP-Gal and β3GalT5, the electron density did not show the presence of galactose (Gal) covalently linked to UDP. This absence is likely due to the hydrolysis of the UDP-Gal donor during crystal formation. To circumvent this issue and obtain structural information on the UDP-Gal binding cleft, we employed an inert analog of the sugar nucleotide donor, UDP-2-deoxy-2-fluoro-α-D-galactopyranose (UDP-2FGal) for cocrystallization with β3GalT5. The replacement of Gal with 2FGal stabilized the donor sugar from hydrolysis. (28) In the enzyme-UDP-2FGal complex, the UDP moiety binds with the divalent ion Mn2+ and the side chain of Tyr128, Lys154, Asp156 (Asp from the DxD motif), and Glu242 (xED motif commonly observed in inverting galactosyltransferase) is hydrogen-bonded with the 2FGal moiety with Tyr128 engaged in additional interaction with the O1 (Figure 2B). These interactions force the 2FGal moiety to place directly above the pyrophosphate moiety in the tucked conformation required for catalytic activity. (31) The side chain of 2FGal was held in the trans–gauche (tg) conformation consistent with the pattern for a β-galactosyltransferases. (32) The donor binding cleft contains Asp158 in the Asp-Ser-Asp (DxD motif) triplet and His285, which is used to coordinate with the diphosphate of UDP together with two water molecules through a Mn2+ divalent cation in an octahedral geometry. The DxD motif is commonly observed in metal-dependent GT-A fold enzyme (33) (e.g., DSD for β3GalT5, DDD for β3GNT2, (25,26) Mfng, (29) and C1GalT1, (28) and DVD for α3GalT (30)). For the UDP moiety, the side chain of Asp126 and Lys134 interacted with the uracil N3 and O2 atoms, respectively; whereas the side chain of Thr64, Gln69, and Ser157 was hydrogen-bonded to the ribose hydroxyls. The side chain of Tyr128 and Lys197 interacted with the pyrophosphate group of UDP (Figure 2B). Specifically, Lys197 was at the long loop (Lys172-Thr217) between β6 and β7, its side chain formed a salt bridge with the α-phosphate oxygen atom, and the main chain interacted with the side chain of Tyr129 at α3. These interactions provided a significant contribution to keep the loop closed, thus facilitating further interaction between Trp198 NE1 and the hydroxyl group on the side chain of Tyr128 to form the acceptor binding cleft for UDP-Gal-Mn2+ (Figure 2D). This strategically arranged loop can prevent excess water from reaching the enzyme’s active site and creates an environment to lower the transition state energy in catalysis and to form a lid over the nucleotide binding site allowing acceptor binding and facilitating the reaction. (34,35)

Figure 2

Figure 2. Donor and acceptor binding clefts in β3GalT5. (A) Surface representation of the β3GalT5 structure, showing the substrate-binding cleft with donor on the left-hand side and acceptor on the right-hand side. (B) Binding cleft for UDP-2FGal and the interacting residues. The left figure shows UDP interactions, and the right figure shows 2F-galactose interactions. Hydrogen bonds are represented by dotted lines. UDP is colored brown and galactose colored gold. (C) Binding cleft for acceptor Gb4 glycan and interacting residues is shown. Hydrogen bonds are represented by dotted lines. Gb4 glycan is colored hot pink. (D) Residues K197 and W198 on the long loop between β6 and β7 sheets interact with residues Y128 and Y129 on α3 helix that form the acceptor binding cleft. The long loop is colored based on residue’s Cα B-factor (average values of 16.76 Å2) with blue to red. The Cα B-factors are depicted on the whole protein structure in blue (lowest B-factor, 8.03 Å2) to red (highest B-factor, 104.36 Å2).

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For the acceptor binding cleft, we observed that β3GalT5 only interacted with the nonreducing end N-acetylgalactosamine (GalNAc) and the following galactose from the structure of β3GalT5 in complex with UDP-Gal and Gb4 glycan. The acceptor GalNAc is stacked against the side chains of Trp198 and Phe213, which placed the acceptor sugar into the catalytic binding cleft. The residue Asp243 (xED motif) further interacted with 3-OH and 4-OH of the acceptor GalNAc. This position structurally aligns well with other GT-A fold enzymes (e.g., D333 in β3GNT2, (25,26) D232 in Mfng, (29) D255 in C1GalT1, (28) D335 in GfsA, and α3GalT, which is switched to xDE (30)). Ser215 formed a hydrogen bond with the acetamide carbonyl, and Glu188 and Tyr210 formed a water-mediated hydrogen bond interaction with the 6-OH of GalNAc. Lys185 and Glu188 were the only two residues that interacted with the following galactose (Figure 2C). The acceptor binding moiety has relatively fewer interactions in comparison to the donor binding moiety, resulting in a wider tolerance to different acceptors (Figure S1). This is in line with our kinetic study, in which the binding of UDP-Gal was around 12-fold stronger than the binding of Gb4 glycan.

Substrate Specificity

While β3GalT5 selectively binds to UDP-Gal as the donor substrate (Figure S4), it exhibits broader selectivity for acceptor substrates (Figure S1). Specifically, β3GalT5 catalyzes the transfer of galactose from UDP-Gal to both the Gb4 glycan (with GalNAc at the nonreducing end) and Lc3 glycan (with GlcNAc at the nonreducing end) via a β1,3-linkage. Using disaccharides as substrates and measuring the end point UDP concentration after 1 h reaction time, we have selected Man-β1,6-Man and GlcNAc-β1,3-GalNAc-α-Thr in addition to Gb4 glycan and GlcNAc-β1,3-Gal-OMe for further structural studies of the enzyme–substrate complex obtained from β3GalT5 cocrystallized with UDP-Gal and soaked with various acceptors. The density maps for the substrates are shown in Figure S6.
The distinction between GalNAc and GlcNAc lies in the position of the 4-OH group. In the ternary structure of β3GalT5:UDP:GlcNAc-β1,3-Gal-OMe (a disaccharide derived from Lc3), the nonreducing end GlcNAc exhibits interactions similar to those observed with GalNAc. Despite the difference in the 4-OH position, the amino acid Asp243 maintains hydrogen bonding with both the 3-OH (2.57 Å for GlcNAc and 2.89 Å for GalNAc) and 4-OH (2.72 Å for GlcNAc and 2.76 Å for GalNAc) groups. The distances between GlcNAc and Asp243 are shorter, potentially resulting in a lower Km value (0.5 mM for GlcNAc-β1,3-Gal-OMe and 2.0 mM for Gb4 glycan). Furthermore, the adjacent galactose residue also engages in comparable interactions. Hence, there are no significant differences in structural conformation (rmsd of 0.10 Å for 234 Cα-atoms) (Figures 3A and S5A,B).

Figure 3

Figure 3. Wide-spectrum acceptor substrate tolerance of β3GalT5. The structures are obtained by cocrystallizing β3GalT5 with UDP-Gal, followed by soaking with different acceptors. (A) Superimposition of Gb4 glycan (in pink; with only the terminal disaccharides for comparison) and the disaccharide GlcNAc-β1,3-Gal of Lc3 (in purple) ligand-bound structures reveals similar interactions. (B) GlcNAc-β1,3-GalNAc-α-Thr (in orange) acceptor binding cleft exhibits identical interactions when compared to the Gb4 glycan bounded structure. (C) Man-β1,6-Man (in green) acceptor ligand-bound structure indicates the formation of the product Gal-β1,3-Man-β1,6-Man. The interacting residues are identical to those observed in other acceptor bound structures with flexible substituents at C2 and C4 and additional interactions with water and product. Hydrogen bonds are represented by dotted lines, and sugars are labeled. The omit maps are shown in Figure S6.

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The discovery of Man-β1,6-Man and GlcNAc-β1,3-GalNAc-α-Thr as substrates with comparable end point UDP concentration after 1 h reaction with Gb4 and Lc3 glycans is intriguing. Notably, the interaction pattern between β3GalT5 and GlcNAc-β1,3-GalNAc-α-Thr mirrors that observed in interactions with Lc3 glycan (Figure 3B). There are no direct interactions between β3GalT5 and the 2-OH group of galactose in the disaccharides of Gb4 or Lc3. Since the galactose moiety protrudes into the open space, its replacement with GalNAc-α-Thr is unlikely to significantly impact the overall structure (with an rmsd of 0.14 Å for 229 Cα-atoms) (Figure S5A). The Km value for GlcNAc-β1,3-GalNAc-Thr is 0.75 mM, similar to that for GlcNAc-β1,3-Gal-OMe (Figure S5B).
Surprisingly, we observed comparable enzyme end point activity for the mannose disaccharide with an β1,6-linkage, and we detected a product with galactose transferred onto the substrate. Notably, there is no significant difference in protein levels when comparing the Gb4 glycan and Man-β1,6-Man-bound structures (with an rmsd of 0.15 Å for 238 Cα-atoms). The dissimilarities at the terminal end of mannose involve the glycosidic bond between 3-OH and galactose, water-mediated interactions between Ser215 and 2-OH of mannose and galactose, and the absence of a water-mediated interaction between 6-OH and Glu188. For the reducing end mannose, the 2-OH interacts with Lys185 and Glu188 interacts with 1-OH and O5 (Figure 3C). Consequently, Man-β1,6-Man exhibits more water-mediated interactions and fewer amino acid direct interactions. Additionally, the β1,6-glycosidic bond between mannose may also contribute to the recognition of such a disaccharide substrate. Further kinetic analysis revealed that the Km value for the Man-β1,6-Man is 2.5 mM, which is the highest among the four substrates and correlates with the structural observation (Figure S5B).

Mechanism of β3GalT5 Inverting Galactosyltransferase

The glycosylation reactions, which involve the nucleophilic substitution of a leaving group in a glycosyl donor with a glycosyl acceptor, can occur via an SN2 or SN2-like mechanism, a stepwise SN1 or SN1-like mechanism involving oxocarbenium intermediates, or SNi mechanisms involving tightly or loosely associated ion pairs, where nucleophilic attack occurs at the same face as the leaving group. Most inverting glycosyltransferases (GTs) are suggested to utilize a displacement mechanism with an oxocarbenium ion-like transition state that happens concurrently in an SN2-like reaction. In contrast, glycosyl oxocarbenium-like intermediates in a double displacement mechanism are plausible for those enzymes that operate through SN1 and SNi mechanisms. It often involves the formation and subsequent breakdown of a covalent glycosyl–enzyme intermediate in which a nucleophile is required for attack on the anomeric center of the donor sugar to form the glycosyl–enzyme species. (36−42)
In this study, we determined three additional ternary structures of β3GalT5. First, in the absence of a glycan acceptor, we observed the exocyclic C1″–O1″ bond breaking in β3GalT5 cocrystallized with UDP-Gal. Second, we observed donor galactose departing from UDP in β3GalT5 cocrystallized with UDP-Gal and soaked with Gb4 glycan. Finally, we investigated β3GalT5 cocrystallized with UDP and soaked with the product Gb5 (SSEA3) glycan. These structures provide insights into the mechanism of the enzymatic reaction. In addition to the structural results, phosphorus-31 NMR spectroscopy shows galactose dissociation at the starting point when the reaction begins to occur (Figures 4E and S7). Thus, we propose that the catalytic mechanism of β3GalT5 involves steps (Figure 4F) diverging from other inverting galactosyltransferases under certain conditions and different from the double displacements proposed for retaining glycosyltransferases. (33,43)

Figure 4

Figure 4. Proposed glycosylation mechanism catalyzed by β3GalT5. (A) β3GalT5 cocrystallized with the UDP-2FGal structure represents the interacting groups in the purposed mechanism (PDB: 8ZWR). (B) β3GalT5 cocrystallized with the UDP-Gal structure represents the purposed mechanism where the glycosidic bond between UDP and galactose is being cleaved (PDB: 8ZX9). (C) β3GalT5:UDP-Gal:Gb4 glycan ternary structure represents the purposed mechanism where Gb4 glycan bound to the acceptor binding cleft and UDP-Gal is in the donor cleft. The departure of UDP is assisted by Mn2+ as a Lewis acid and the phenolic group from Tyr-128 as a general acid, leading to a partial interaction of the phenolic oxygen with the anomeric carbon. The acceptor hydroxyl group serves as a nucleophile assisted by the Asp-243 carboxylate as a general base (PDB: 8ZX8). (D) β3GalT5:UDP-Gal:Gb5 glycan ternary structure represents the proposed product formation (PDB: 8ZWW). (E) P-31 signals of UDP, UDP-Gal, β3GalT5 with UDP-Gal, and β3GalT5 with UDP-Gal and Gb4 glycan at the beginning of mixing. Red arrows indicate the P-31 signal of UDP, indicating the enzymatic reaction of UDP-Gal with water as an acceptor is much slower than that with Gb4 glycan as an acceptor. (F) Proposed SN2-like mechanisms for β3GalT5-catalyzed hydrolysis of UDP-Gal in the absence of a glycan acceptor to generate galactopyranose. The activated UDP-Gal with oxocarbenium character may collapse to another product through an SN1-like mechanism (see the discussion). (G) Proposed SN2-like mechanism for reaction with the glycan acceptor.

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Initially, the glycosidic bond between UDP and the donor galactose is partially broken yet remains within an interacting distance range (approximately 3.34 Å), (Figure 4B). In the enzymatic glycosyl transfer reaction, a metal ion and/or general acid assistance is required to facilitate the cleavage of the exocyclic C1″–O1″ bond. In this case, the departure of the leaving group UDP is assisted by Mn2+ as a Lewis acid and the side chain hydroxyl of Tyr128 (2.67 Å) as a general acid (Figure 4B), which initially interacts with both C1″, O1″, and O5″ at distances of 3.31, 3.45, and 3.01 Å, respectively (Figure 4A). However, in the absence of a glycan acceptor, we observed a slow breakdown of UDP-Gal to galactose and another product with electron density such as the oxocarbenium intermediate, presumably a stable structure collapsed from the high-energy oxocarbenium intermediate. Apparently, a water molecule in the active site acts as an acceptor to generate the galactose product (Figure 4F), and this process is assisted by the carboxylate of Asp243 as a general base (Figure S8) to facilitate hydrolysis. The water molecule can also interact with the partially positive charge O5 of the oxocarbenium-like high-energy intermediate and this is consistent with the QM/MM calculations of Thermus thermophilus β-glycosidase showing a neighboring water molecule interacting with the oxocarbenium-like intermediate through O5. (44) Tyr128 and Lys197 further interact with the diphosphate of UDP to facilitate the departure of UDP and the formation of galactopyranose (Figure 4A,B). This background hydrolysis of the donor substrate is often observed in GTs, including β3GalT5, which explains why we observed the dissociation of donor galactose in the absence of an acceptor and at the starting point of the reaction when an acceptor was added. (34,45−47)
In the glycosyl transfer reaction, Asp243 acts as a general base, deprotonating the glycan acceptor 3-OH on the GalNAc (2.32 Å) for a nucleophilic reaction with the anomeric carbon of the galactose (2.71 Å) (Figure 4C), resulting in the formation of a new β-1,3-glycosidic bond between the galactose and the GalNAc (Figure 4D). Our experimental method enabled us to observe these results, likely due to the initial cocrystallization of β3GalT5 with UDP-Gal followed by soaking with the acceptor. This soaking experiment allowed us to capture the breaking of the UDP-Gal bond and the formation of the new glycosylic bond with the acceptor. Collectively, the β3GalT5-catalyzed glycosyl transfer reaction utilizes an SN2-type reaction (Figure 4F,G) and more likely a nonconcerted SN2-like mechanism. In the enzymatic reaction with weak acceptors like water, the oxocarbenium-like high-energy intermediate may react with water to form galactopyranose through an SN2-like mechanism or collapse to a stable product with a similar electron density through an SN1-like mechanism. Based on our observations, the enzymatic reaction mechanism appears to be dependent on the reactivity of a bound acceptor. However, for a comprehensive understanding of the reaction with different acceptors, further quantitative analysis and kinetic studies, coupled with improved structural resolution, are necessary to elucidate the details of this dynamic enzymatic reaction at the atomic level.
Even though there are no major conformational changes at the protein level, the side chains of Tyr128, Asp156, Lys197, and Trp198 exhibit significant alterations during the enzymatic reaction (Figures 4A–D and S9–S11). First, the side chain of Tyr128 moves toward the leaving group, UDP, throughout the reaction events, maintaining an approximate distance of 1.09 Å (Figure S9). Second, Asp156 plays a crucial role in transferring the donor galactose to the acceptor. It interacts with the C-3 hydroxyl group on the galactose, with a movement of approximately 0.52 Å toward the acceptor when galactose dissociates from UDP. Upon acceptor binding, this movement increases to 0.84 Å. Interestingly, when the product Gb5 glycan is formed, Asp156 returns to a position close to that observed in the UDP-2FGal bound structure (Figure S10). Third, the side chain of Lys197 stabilizes the UDP leaving group by shifting its interactions from α-phosphate to β-phosphate (Figure 4A–D). Finally, the side chain of Trp198 exhibits dynamic “lid on” and “lid off” conformational change. The angle difference between the initial UDP-2FGal bound structure and the final product bound structure is approximately 33°. The indole lid is turned off when no receptor is bound but is activated by forming a stacking interaction with the nonreducing end of acceptor (Figure S11). In our mutagenesis study, we observed abolished enzymatic activity through mutating the key residues interacting with donor galactose, divalent ion Mn2+, acceptor Gb4 glycan, and product Gb5 glycan (Figure S12). These observations provide valuable insights into the dynamic behavior of key residues during the β3GalT5-catalyzed glycosylation process.

Conformation of Donor Galactose and Coordination Geometry of the Divalent Ion Mn2+ in Reaction

We observed a distortion in the galactopyranose ring upon binding of the donor, potentially indicating the presence of oxocarbenium-like intermediates. The flattened geometry of the donor galactopyranose ring is also consistent with oxocarbenium-like character. Specifically, a partially positive charge (δ+) was developed on the anomeric carbon, which was stabilized by the phenolic oxygen of Tyr128. This portion of the structure must be planar. Consequently, there are eight possible conformers (4H3, 3H4, 2,5B, B2,5, 4E, E4, 3E, and E3) for stabilizing the pyranose as an oxocarbenium ion state. (48,49) The 1,4B ring conformation has also been reported in inverting galactosyltranserases (50) and glycoside hydrolases. (51) Among these conformers, both the 4H3 and 3H4 half-chair conformations are well-supported as the transition state geometry, (52) and the 1,4B, 2,5B, and B2,5 boat conformations are all identified in the Stoddart diagram. (53) In the complex structure of β3GalT5 with UDP and dissociated galactose, the galactopyranose ring adopted an 4H3 half-chair conformation within the catalytic binding domain. However, in the presence of the Gb4-glycan acceptor, the deprotonated 3-OH of the GalNAc acceptor further interacts with the dissociated donor galactose, which might create a potential force to pull the C1 and the galactose pyranose transitioned to the 1,4B boat conformation, facilitating bond formation. The weak electron density observed between O5 and the anomeric C1 in the galactopyranose ring may result from constant electron shifts between single and partial double bonds. Subsequently, in complex with the Gb5-glycan product, the galactopyranose ring returned to the 4C1 chair conformation. Overall, the conformational changes in the galactopyranose ring followed the sequence: 4C1 → 4H3 → 1,4B → 4C1 with possibility that there might be other intermediates in between where we yet to capture. Although further mechanistic studies with kinetic studies and computational calculations will support our findings, this study represents the first structure-based observation of the oxocarbenium-like intermediate in an inverted galactosyl transferase-catalyzed reaction (Figure 5).

Figure 5

Figure 5. Conformations and electron density maps of donor galactose in enzymatic reaction. The conformations of donor galactose (colored gold) from binding of UDP-Gal to formation of product Gb5 glycan. Side views of galactose show the planar conformation (colored gray). Fo–Fc polder omit electron-density maps are contoured at 4σ. From left to right, PDB: 8ZWR represents donor galactose from UDP2FGal (UDPGal); PDB: 8ZX9 (UDP + Gal) represents dissociated galactose from the structure of enzyme cocrystallized with UDP-Gal; PDB: 8ZX8 (UDP + Gal + Gb4) represents dissociated galactose from the structure of enzyme cocrystallized with UDP-Gal and soaked with Gb4-glycan; and PDB: 8ZWW (UDP + Gb5) represents the galactose from the final product Gb5-glycan where β3GalT5 is cocrystallized with UDP-Gal and soaked with SSEA3-glycan.

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The donor binding cleft contains a “DXD motif” that consists of an Asp-X-Asp triplet used to coordinate the phosphates of the donor molecule through a divalent cation with an octahedral geometry. In our study, the octahedral geometry surrounding the Mn2+ metal is more symmetrical in the UDP-2FGal bound structure with displayed angle ranging from 82.95° to 94.07° and in the Gb5-glycan bound structure with displayed angle ranging from 75.93° to 91.38°. The six bond distances are also more correlated with each other with distances ranging from 2.16 to 2.53 Å for the UDP-2FGal bound structure and 2.14 to 2.49 Å for the Gb5-glycan bound structure. However, when the galactose is in the oxocarbenium-like state, we can observe the distortion with galactose dissociation in the octahedral geometry. In the UDP and Gal bound structure without Gb4-glycan acceptor, the displayed angles range from 82.84° to 99.82° and the bond distances range from 1.97 to 2.69 Å. With Gb4-glycan bound, the octahedral geometry is skewed to nearly trigonal prismatic dimensions with acute bidentate Asp158 coordination employed (displayed angles range from 51.91° to 102.73°) (Figures 6 and S13). The center of the Mn2+ coordinated geometry shifts toward the β-phosphate, allows sufficient room for the attack from the acceptor, and facilitates the departure of the leaving group. (54)

Figure 6

Figure 6. Octahedral geometry of the coordinated divalent ion Mn2+. The Mn2+(colored purple) octahedral binding partners are Asp158 (from the DXD motif), His 285, diphosphate from UDP, and two water molecules (colored red). In the β3GalT5:UDP:Gal:Gb4 structure, the coordinated partners shift from one water molecule to bidentate Asp 158. The density maps for the four structures are shown in Figure S13 of the Supporting Information.

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Conclusions

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We here provided structural insights into the substrate specificity and possible reaction mechanisms of the β3GalT5-catalyzed reaction. This cancer-related enzyme plays a crucial role in glycolipid biosynthesis and demonstrates intriguing substrate preferences. While it selectively binds to UDP-galactose as the donor substrate, it maintains a broader selectivity for acceptor substrates. Specifically, β3GalT5 catalyzes the transfer of galactose from UDP-Gal to both Gb4 glycan (with GalNAc at the nonreducing end) and Lc3 glycan (with GlcNAc at the nonreducing end) via a β1,3-linkage. In our study of substrate specificity involving disaccharides, β3GalT5 also exhibits robust activities toward Mannose-β1,6-Mannose and GlcNAc-β1,3-GalNAc-α-Thr in addition to Gb4 and Lc3 glycans. We illustrate the key interactions in the glycosylation processes with different acceptor substrates based on the structures. Additionally, we uncover the mechanism of β3GalT5 catalysis by solving the structures of different catalysis steps, including the Michaelis complex, the oxocarbenium-like intermediate, and the reaction products. Based on the structures observed in our study, the β3GalT5-catalyzed glycosyl transfer reaction utilizes an SN2-like mechanism, where the donor substrate UDP-Gal is activated by Mn2+ and Tyr-128 to facilitate the departure of UDP and the acceptor hydroxyl group acts as a nucleophile to react with the anomeric carbon of UDP-Gal under the assistance of Asp243 carboxylate as a general base to generate a new glycosidic bond in β1,3-linkage. However, in the enzymatic reaction without a glycan acceptor, our structural studies indicate that UDP-Gal is activated and reacted with water as an acceptor through an SN2-like mechanism to form the galactopyranose product. In addition, the activated UDP-Gal with an oxocarbenium-like structure may collapse through an SN1-like mechanism to form a product with similar electron density to the oxocarbenium intermediate of galactose, likely the enol form of galactal (Figure S14). These observations suggest that the mechanism of the enzymatic reaction may change, depending on the reactivity of the bound acceptor, and could range from SN2-like for reaction with good acceptors to SN1-like mechanism for weak acceptors. It is noted that the proposed mechanisms are based on the structures obtained from our experiments and may not precisely reflect the reactions in the solution. Nevertheless, the broad-spectrum acceptor substrate specificity and the thorough investigation into the structure, function, and mechanism of β3GalT5 not only enhance our understanding of its potential in glycan synthesis but also illuminate its crucial role in cancer biology. The findings offer new opportunities for synthetic glycobiology and drug design, specifically targeting β3GalT5 in diseases.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c11724.

  • Synthetic and experimental procedures, characterization and structural analyses, and crystallographic information (PDF)

Accession Codes

Atomic coordinates and structural factors for the reported 8 crystal structures have been deposited in the Protein Data Bank under the accession number (8ZWR, 8ZWP, 8ZWW, 8ZWY, 8ZX2, 8ZX3, 8ZX8, and 8ZX9). Other data are available from the corresponding author upon reasonable request.

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

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  • Corresponding Authors
  • Authors
    • Jennifer M. Lo - Genomics Research Center, Academia Sinica, Taipei 115, Taiwan;  Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan;  Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan
    • Chih-Chuan Kung - Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
    • Ting-Jen Rachel Cheng - Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. J.M.L. and C.-C.K. contributed equally.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was funded by Academia Sinica: AS-IA-113-L02 (to C.M.). Portions of this research were conducted at the National Synchrotron Radiation Research Center (NSRRC), a national user facility supported by MOST of Taiwan. We thank NSRRC for allocation of beamlines TLS13B, TLS15A, and TPS05A. We also thank Mrs. Yi-Ping Huang for assistance with the NMR analysis and technical support and Mrs. Meng-Chuan Chang for assistance with crystallization screening at Genomics Research Center, Academia Sinica.

References

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    High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner
    Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.
    Journal of Biological Chemistry (2015), 290 (45), 27040-27052CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been detd. to between 1.55 and 1.39 Å resoln. in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems assocd. with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are obsd. to maintain a const. location, the sugar moieties lie in four distinct conformations, varying from extended to the "tucked under" conformation assocd. with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the obsd. conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be "isosteric" with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB.
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    Quirke, J. C. K.; Crich, D. Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases. ACS Catal. 2021, 11 (9), 5069– 5078,  DOI: 10.1021/acscatal.1c00896
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    Moremen, K. W.; Haltiwanger, R. S. Emerging structural insights into glycosyltransferase-mediated synthesis of glycans. Nat. Chem. Biol. 2019, 15 (9), 853– 864,  DOI: 10.1038/s41589-019-0350-2
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    Emerging structural insights into glycosyltransferase-mediated synthesis of glycans
    Moremen, Kelley W.; Haltiwanger, Robert S.
    Nature Chemical Biology (2019), 15 (9), 853-864CODEN: NCBABT; ISSN:1552-4450. (Nature Research)
    A review. Glycans linked to proteins and lipids play key roles in biol.; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures obsd. in biol.
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    Brockhausen, I. Crossroads between Bacterial and Mammalian Glycosyltransferases. Front. Immunol. 2014, 5, 492,  DOI: 10.3389/fimmu.2014.00492
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    Crossroads between Bacterial and Mammalian Glycosyltransferases
    Brockhausen Inka
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    Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.
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    Rini, J. M.; Moremen, K. W.; Davis, B. G. Glycosyltransferases and Glycan-Processing Enzymes. In Essentials of Glycobiology [Internet]; Varki, A.; Cumming, R. D.; Esko, J. D., et al., Eds.; Cold Spring Harbor Laboratory Press, 2022.
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    Chan, J.; Tang, A.; Bennet, A. J. A stepwise solvent-promoted SNi reaction of alpha-D-glucopyranosyl fluoride: mechanistic implications for retaining glycosyltransferases. J. Am. Chem. Soc. 2012, 134 (2), 1212– 1220,  DOI: 10.1021/ja209339j
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    A Stepwise Solvent-Promoted SNi Reaction of α-D-Glucopyranosyl Fluoride: Mechanistic Implications for Retaining Glycosyltransferases
    Chan, Jefferson; Tang, Ariel; Bennet, Andrew J.
    Journal of the American Chemical Society (2012), 134 (2), 1212-1220CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-d-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH⧺ = 81.4 ± 1.7 kJ mol-1, and ΔS⧺ = -90.3 ± 4.6 J mol-1 K-1. To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, resp. The transition state for the solvolysis reaction was modeled computationally using the exptl. KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an SNi (DN⧺*ANss) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol mol. In comparison, the inverted product, 1,6-anhydro-β-d-glucopyranose, is formed by intramol. capture of a solvent-equilibrated glucopyranosylium ion, which results from dissocn. of the solvent-sepd. ion pair formed in the rate-limiting ionization reaction (DN⧺ + AN). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.
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    Cowdrey, W. A.; Hughes, E. D.; Ingold, C. K.; Masterman, S.; Scott, A. D. Reaction kinetics and the Walden inversion Part VI Relation of steric orientation to mechanism in substitution involving halogen atoms and simple or substituted hydroxyl groups. Journal of the Chemical Society 1937, 0, 1252– 1271,  DOI: 10.1039/jr9370001252
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    Crich, D. Mechanism of a chemical glycosylation reaction. Acc. Chem. Res. 2010, 43 (8), 1144– 1153,  DOI: 10.1021/ar100035r
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    Mechanism of a Chemical Glycosylation Reaction
    Crich, David
    Accounts of Chemical Research (2010), 43 (8), 1144-1153CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Glycosylation is arguably the most important reaction in the field of glycochem., yet it involves one of the most empirically interpreted mechanisms in the science of org. chem. The β-mannopyranosides, long considered one of the more difficult classes of glycosidic bond to prep., were no exception to this rule. Several logical but circuitous routes for their prepn. were described in the literature, but they were accompanied by a greater no. of mostly ineffective recipes for their direct access. This situation changed in 1996 with the discovery of the 4,6-O-benzylidene acetal as a control element permitting direct entry into the β-mannopyranosides, typically with high yield and selectivity. The unexpected nature of this phenomenon demanded study of the mechanism, leading first to the demonstration of the α-mannopyranosyl triflates as reaction intermediates and then to the development of α-deuterium kinetic isotope effect methods to probe their transformation into the product glycosides. In this account, the authors assemble their observations into a comprehensive assessment consistent with a single mechanistic scheme. The realization that in the glucopyranose series the 4,6-O-benzylidene acetal is α- rather than β-directing led to further investigations of substituent effects on the stereoselectivity of these glycosylation reactions, culminating in their explanation in terms of the covalent α-glycosyl triflates acting as a reservoir for a series of transient contact and solvent-sepd. ion pairs. The function of the benzylidene acetal, as explained by Bols and co-workers, is to lock the C6-O6 bond antiperiplanar to the C5-O5 bond, thereby maximizing its electron-withdrawing effect, destabilizing the glycosyl oxocarbenium ion, and shifting the equil. as far as possible toward the covalent triflate. β-Selective reactions result from attack of the nucleophile on the transient contact ion pair in which the α-face of the oxocarbenium ion is shielded by the triflate counterion. The α-products arise from attack either on the solvent-sepd. ion pair or on a free oxocarbenium ion, according to the dictates of the anomeric effect. Changes in selectivity from varying stereochem. (glucose vs. mannose) or from using different protecting groups can be explained by the shifting position of the key equil. and, in particular, by the energy differences between the covalent triflate and the ion pairs. Of particular note is the importance of substituents at the 3-position of the donor; an explanation is proposed that invokes their evolving torsional interaction with the substituent at C2 as the chair form of the covalent triflate moves toward the half-chair of the oxocarbenium ion.
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    Fu, Y.; Bernasconi, L.; Liu, P. Ab Initio Molecular Dynamics Simulations of the S(N)1/S(N)2 Mechanistic Continuum in Glycosylation Reactions. J. Am. Chem. Soc. 2021, 143 (3), 1577– 1589,  DOI: 10.1021/jacs.0c12096
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    Iglesias-Fernandez, J.; Hancock, S. M.; Lee, S. S.; Khan, M.; Kirkpatrick, J.; Oldham, N. J.; McAuley, K.; Fordham-Skelton, A.; Rovira, C.; Davis, B. G. A front-face ‘S(N)i synthase’ engineered from a retaining ‘double-S(N)2’ hydrolase. Nat. Chem. Biol. 2017, 13 (8), 874– 881,  DOI: 10.1038/nchembio.2394
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    Lee, S. S.; Hong, S. Y.; Errey, J. C.; Izumi, A.; Davies, G. J.; Davis, B. G. Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase. Nat. Chem. Biol. 2011, 7 (9), 631– 638,  DOI: 10.1038/nchembio.628
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    Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
    Lee, Seung Seo; Hong, Sung You; Errey, James C.; Izumi, Atsushi; Davies, Gideon J.; Davis, Benjamin G.
    Nature Chemical Biology (2011), 7 (9), 631-638CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
    A previously detd. crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group 18O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Bronsted anal. of nucleophiles and Taft anal. highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quant. data substantiate this unusual enzymic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.
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    Paparella, A. S.; Cahill, S. M.; Aboulache, B. L.; Schramm, V. L. Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an S(N)i Mechanism and Ion Pair Transition State. ACS Chem. Biol. 2022, 17 (9), 2507– 2518,  DOI: 10.1021/acschembio.2c00408
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    Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an SNi Mechanism and Ion Pair Transition State
    Paparella, Ashleigh S.; Cahill, Sean M.; Aboulache, Briana L.; Schramm, Vern L.
    ACS Chemical Biology (2022), 17 (9), 2507-2518CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    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 stereochem. 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 stereochem., while hydrolysis of UDP-glucose by TcdB causes inversion of stereochem. Kinetic anal. revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an SNi mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mech. calcns. revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an SNi 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.
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    Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 2008, 77 (0066–4154 (Print)), 521– 555. DOI: 10.1146/annurev.biochem.76.061005.092322
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    Glycosyltransferases: structures, functions, and mechanisms
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    A review. Glycosyltransferases catalyze glycosidic bond formation using sugar donors contg. a nucleoside phosphate or a lipid phosphate leaving group. Only 2 structural folds, GT-A and GT-B, have been identified for the nucleotide sugar-dependent enzymes, but other folds are now appearing for the sol. domains of lipid phosphosugar-dependent glycosyltransferases. Structural and kinetic studies have provided new insights. Inverting glycosyltransferases utilize a direct displacement SN2-like mechanism involving an enzymic base catalyst. Leaving group departure in GT-A fold enzymes is typically facilitated via a coordinated divalent cation, whereas GT-B fold enzymes instead use pos. charged side-chains and/or hydroxyls and helix dipoles. The mechanism of retaining glycosyltransferases is less clear. The expected 2-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected. A mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
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    Original Research

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    Bowles, W. H. D.; Gloster, T. M. Sialidase and Sialyltransferase Inhibitors: Targeting Pathogenicity and Disease. Front. Mol. Biosci. 2021, 8, 705133  DOI: 10.3389/fmolb.2021.705133
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    Sialidase and sialyltransferase inhibitors: targeting pathogenicity and disease
    Bowles, William H. D.; Gloster, Tracey M.
    Frontiers in Molecular Biosciences (2021), 8 (), 705133CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
    A review. Sialidases (SAs) and sialyltransferases (STs), the enzymes responsible for removing and adding sialic acid to other glycans, play essential roles in viruses, bacteria, parasites, and humans. Sialic acid is often the terminal sugar on glycans protruding from the cell surface in humans and is an important component for recognition and cell function. Pathogens have evolved to exploit this and use sialic acid to either "cloak" themselves, ensuring they remain undetected, or as a mechanism to enable release of virus progeny. The development of inhibitors against SAs and STs therefore provides the opportunity to target a range of diseases. Inhibitors targeting viral, bacterial, or parasitic enzymes can directly target their pathogenicity in humans. Excellent examples of this can be found with the anti-influenza drugs Zanamivir (Relenza, GlaxoSmithKline) and Oseltamivir (Tamiflu, Roche and Gilead), which have been used in the clinic for over two decades. However, the development of resistance against these drugs means there is an ongoing need for novel potent and specific inhibitors. Humans possess 20 STs and four SAs that play essential roles in cellular function, but have also been implicated in cancer progression, as glycans on many cancer cells are found to be hyper-sialylated. While much remains unknown about how STs function in relation to disease, it is clear that specific inhibitors of them can serve both as tools to gain a better understanding of their activity and form the basis for development of anti-cancer drugs. Here we review the recent developments in the design of SA and ST inhibitors against pathogens and humans.
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    Mehr, K.; Withers, S. G. Mechanisms of the sialidase and trans-sialidase activities of bacterial sialyltransferases from glycosyltransferase family 80. Glycobiology 2016, 26 (4), 353– 359,  DOI: 10.1093/glycob/cwv105
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    Sugiarto, G.; Lau, K.; Qu, J.; Li, Y.; Lim, S.; Mu, S.; Ames, J. B.; Fisher, A. J.; Chen, X. A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating LewisX. ACS Chem. Biol. 2012, 7 (7), 1232– 1240,  DOI: 10.1021/cb300125k
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    A Sialyltransferase Mutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities for Directly Sialylating Lewisx
    Sugiarto, Go; Lau, Kam; Qu, Jingyao; Li, Yanhong; Lim, Sunghyuk; Mu, Shengmao; Ames, James B.; Fisher, Andrew J.; Chen, Xi
    ACS Chemical Biology (2012), 7 (7), 1232-1240CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Glycosyltransferases are important catalysts for enzymic and chemoenzymic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-contg. acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymic synthesis of complex sialyl Lewisx antigens contg. different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.
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    Nin-Hill, A.; Rovira, C. The Catalytic Reaction Mechanism of the β-Galactocerebrosidase Enzyme Deficient in Krabbe Disease. ACS Catal. 2020, 10 (20), 12091– 12097,  DOI: 10.1021/acscatal.0c02609
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    Franconetti, A.; Arda, A.; Asensio, J. L.; Bleriot, Y.; Thibaudeau, S.; Jimenez-Barbero, J. Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions. Acc. Chem. Res. 2021, 54 (11), 2552– 2564,  DOI: 10.1021/acs.accounts.1c00021
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    Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions
    Franconetti, Antonio; Arda, Ana; Asensio, Juan luis; Bleriot, Yves; Thibaudeau, Sebastien; Jimenez-barbero, Jesus
    Accounts of Chemical Research (2021), 54 (11), 2552-2564CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
    A review. Carbohydrates (glycans, saccharides, sugars) are essential mols. in all domains of life. Research on glycoscience spans from chem. to biomedicine, including material science and biotechnol. The access to pure and well-defined complex glycans using synthetic methods depends on the success of the employed glycosylation reaction. In most cases, the mechanism of the glycosylation reaction is supposed to involve the oxocarbenium ion. Understanding the structure, conformation, reactivity, and interactions of these glycosyl cations is essential to predict the outcome of the reaction. In this Account, building on our contributions on this topic, we discuss the theor. and exptl. approaches that have been employed to decipher the key features of glycosyl cations, from their structures to their interactions and reactivity. We also highlight that, from a chem. perspective, the glycosylation reaction can be described as a continuum, from unimol. SN1 with naked oxocarbenium cations as intermediates to bimol. SN2-type mechanisms, which involve the key role of counterions and donors. All these factors should be considered and are discussed herein. The importance of dissociative mechanisms (involving contact ion pairs, solvent-sepd. ion pairs, solvent-equilibrated ion pairs) with bimol. features in most reactions is also highlighted. The role of theor. calcns. to predict the conformation, dynamics and reactivity of the oxocarbenium ion is also discussed, highlighting the advances in this field that now allow the access to the conformational preferences of a variety of oxocarbenium ions and their reactivities under SN1-like conditions. Specifically, the ground-breaking use of superacids to generate these cations is emphasized, since it has permitted characterizing the structure and conformation of a variety of glycosyl oxocarbenium ions in superacid soln. by NMR spectroscopy. We also pay special attention to the reactivity of these glycosyl ions that depends on the conditions, including the counterions, the possible intra- or intermol. participation of functional groups that may stabilize the cation and the chem. nature of the acceptor, either weak or strong nucleophile. We discuss recent investigations from different exptl. perspectives, which identified the involved ionic intermediates, estg. their lifetimes and reactivities and studying their interactions with other mols. In this context, we also emphasize the relationship between the chem. methods that can be employed to modulate the sensitivity of glycosyl cations and the way in which glycosyl modifying enzymes (glycosyl hydrolases and transferases) build and cleave glycosidic linkages in nature. This comparison provides inspiration on the use of mols. that regulate the stability and reactivity of glycosyl cations.
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    Darby, J. F.; Gilio, A. K.; Piniello, B.; Roth, C.; Blagova, E.; Hubbard, R. E.; Rovira, C.; Davies, G. J.; Wu, L. Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V. ACS Catal. 2020, 10 (15), 8590– 8596,  DOI: 10.1021/acscatal.0c02222
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    Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V
    Darby, John F.; Gilio, Amelia K.; Piniello, Beatriz; Roth, Christian; Blagova, Elena; Hubbard, Roderick E.; Rovira, Carme; Davies, Gideon J.; Wu, Liang
    ACS Catalysis (2020), 10 (15), 8590-8596CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
    α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. The authors solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-assocd. N-glycosylation.
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    Catalytic Itinerary in 1,3-1,4-β-Glucanase Unraveled by QM/MM Metadynamics. Charge Is Not Yet Fully Developed at the Oxocarbenium Ion-like Transition State
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    Journal of the American Chemical Society (2011), 133 (50), 20301-20309CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Retaining glycoside hydrolases (GHs), key enzymes in the metab. of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a 1,4B/1S3 conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction (1,4B/1S3 -4E/4H3 - 4C1) and shows that the transition state is not the point of max. charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.
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Published March 25, 2025

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  • Abstract

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    Figure 1

    Figure 1. Overall structure of human β3GalT5. (A) Synthesis of SSEA-3 from Gb4 catalyzed by β3GalT5. (B) β3GalT5 catalyzed galactosylation in the synthesis of Core 3 O-glycans, globo-series glycosphingolipids (GSLs), and lacto-series GSLs. (C) Construct of gene for β3GalT5 expression. (D) Overall structure of β3GalT5 with substrates: composite image with β3GalT5:UDP2FGal superimposed on β3GalT5:Gb4 glycan. The color gradient from light to dark blue indicates the protein structure from N-terminal to C-terminal. The divalent ion Mn2+ is colored purple, UDP is brown, donor sugar galactose is gold, and acceptor Gb4 glycan is green.

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    Figure 2

    Figure 2. Donor and acceptor binding clefts in β3GalT5. (A) Surface representation of the β3GalT5 structure, showing the substrate-binding cleft with donor on the left-hand side and acceptor on the right-hand side. (B) Binding cleft for UDP-2FGal and the interacting residues. The left figure shows UDP interactions, and the right figure shows 2F-galactose interactions. Hydrogen bonds are represented by dotted lines. UDP is colored brown and galactose colored gold. (C) Binding cleft for acceptor Gb4 glycan and interacting residues is shown. Hydrogen bonds are represented by dotted lines. Gb4 glycan is colored hot pink. (D) Residues K197 and W198 on the long loop between β6 and β7 sheets interact with residues Y128 and Y129 on α3 helix that form the acceptor binding cleft. The long loop is colored based on residue’s Cα B-factor (average values of 16.76 Å2) with blue to red. The Cα B-factors are depicted on the whole protein structure in blue (lowest B-factor, 8.03 Å2) to red (highest B-factor, 104.36 Å2).

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    Figure 3

    Figure 3. Wide-spectrum acceptor substrate tolerance of β3GalT5. The structures are obtained by cocrystallizing β3GalT5 with UDP-Gal, followed by soaking with different acceptors. (A) Superimposition of Gb4 glycan (in pink; with only the terminal disaccharides for comparison) and the disaccharide GlcNAc-β1,3-Gal of Lc3 (in purple) ligand-bound structures reveals similar interactions. (B) GlcNAc-β1,3-GalNAc-α-Thr (in orange) acceptor binding cleft exhibits identical interactions when compared to the Gb4 glycan bounded structure. (C) Man-β1,6-Man (in green) acceptor ligand-bound structure indicates the formation of the product Gal-β1,3-Man-β1,6-Man. The interacting residues are identical to those observed in other acceptor bound structures with flexible substituents at C2 and C4 and additional interactions with water and product. Hydrogen bonds are represented by dotted lines, and sugars are labeled. The omit maps are shown in Figure S6.

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    Figure 4

    Figure 4. Proposed glycosylation mechanism catalyzed by β3GalT5. (A) β3GalT5 cocrystallized with the UDP-2FGal structure represents the interacting groups in the purposed mechanism (PDB: 8ZWR). (B) β3GalT5 cocrystallized with the UDP-Gal structure represents the purposed mechanism where the glycosidic bond between UDP and galactose is being cleaved (PDB: 8ZX9). (C) β3GalT5:UDP-Gal:Gb4 glycan ternary structure represents the purposed mechanism where Gb4 glycan bound to the acceptor binding cleft and UDP-Gal is in the donor cleft. The departure of UDP is assisted by Mn2+ as a Lewis acid and the phenolic group from Tyr-128 as a general acid, leading to a partial interaction of the phenolic oxygen with the anomeric carbon. The acceptor hydroxyl group serves as a nucleophile assisted by the Asp-243 carboxylate as a general base (PDB: 8ZX8). (D) β3GalT5:UDP-Gal:Gb5 glycan ternary structure represents the proposed product formation (PDB: 8ZWW). (E) P-31 signals of UDP, UDP-Gal, β3GalT5 with UDP-Gal, and β3GalT5 with UDP-Gal and Gb4 glycan at the beginning of mixing. Red arrows indicate the P-31 signal of UDP, indicating the enzymatic reaction of UDP-Gal with water as an acceptor is much slower than that with Gb4 glycan as an acceptor. (F) Proposed SN2-like mechanisms for β3GalT5-catalyzed hydrolysis of UDP-Gal in the absence of a glycan acceptor to generate galactopyranose. The activated UDP-Gal with oxocarbenium character may collapse to another product through an SN1-like mechanism (see the discussion). (G) Proposed SN2-like mechanism for reaction with the glycan acceptor.

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    Figure 5

    Figure 5. Conformations and electron density maps of donor galactose in enzymatic reaction. The conformations of donor galactose (colored gold) from binding of UDP-Gal to formation of product Gb5 glycan. Side views of galactose show the planar conformation (colored gray). Fo–Fc polder omit electron-density maps are contoured at 4σ. From left to right, PDB: 8ZWR represents donor galactose from UDP2FGal (UDPGal); PDB: 8ZX9 (UDP + Gal) represents dissociated galactose from the structure of enzyme cocrystallized with UDP-Gal; PDB: 8ZX8 (UDP + Gal + Gb4) represents dissociated galactose from the structure of enzyme cocrystallized with UDP-Gal and soaked with Gb4-glycan; and PDB: 8ZWW (UDP + Gb5) represents the galactose from the final product Gb5-glycan where β3GalT5 is cocrystallized with UDP-Gal and soaked with SSEA3-glycan.

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    Figure 6

    Figure 6. Octahedral geometry of the coordinated divalent ion Mn2+. The Mn2+(colored purple) octahedral binding partners are Asp158 (from the DXD motif), His 285, diphosphate from UDP, and two water molecules (colored red). In the β3GalT5:UDP:Gal:Gb4 structure, the coordinated partners shift from one water molecule to bidentate Asp 158. The density maps for the four structures are shown in Figure S13 of the Supporting Information.

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      Brockhausen Inka
      Frontiers in immunology (2014), 5 (), 492 ISSN:1664-3224.
      Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen-Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures.
    35. 35
      Rini, J. M.; Moremen, K. W.; Davis, B. G. Glycosyltransferases and Glycan-Processing Enzymes. In Essentials of Glycobiology [Internet]; Varki, A.; Cumming, R. D.; Esko, J. D., et al., Eds.; Cold Spring Harbor Laboratory Press, 2022.
      There is no corresponding record for this reference.
    36. 36
      Chan, J.; Tang, A.; Bennet, A. J. A stepwise solvent-promoted SNi reaction of alpha-D-glucopyranosyl fluoride: mechanistic implications for retaining glycosyltransferases. J. Am. Chem. Soc. 2012, 134 (2), 1212– 1220,  DOI: 10.1021/ja209339j
      36
      A Stepwise Solvent-Promoted SNi Reaction of α-D-Glucopyranosyl Fluoride: Mechanistic Implications for Retaining Glycosyltransferases
      Chan, Jefferson; Tang, Ariel; Bennet, Andrew J.
      Journal of the American Chemical Society (2012), 134 (2), 1212-1220CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The solvolysis of α-d-glucopyranosyl fluoride in hexafluoro-2-propanol gives two products, 1,1,1,3,3,3-hexafluoropropan-2-yl α-d-glucopyranoside and 1,6-anhydro-β-d-glucopyranose. The ratio of these two products is essentially unchanged for reactions that are performed between 56 and 100 °C. The activation parameters for the solvolysis reaction are as follows: ΔH⧺ = 81.4 ± 1.7 kJ mol-1, and ΔS⧺ = -90.3 ± 4.6 J mol-1 K-1. To characterize, by use of multiple kinetic isotope effect (KIE) measurements, the TS for the solvolysis reaction in hexafluoro-2-propanol, we synthesized a series of isotopically labeled α-d-glucopyranosyl fluorides. The measured KIEs for the C1 deuterium, C2 deuterium, C5 deuterium, anomeric carbon, ring oxygen, O6, and solvent deuterium are 1.185 ± 0.006, 1.080 ± 0.010, 0.987 ± 0.007, 1.008 ± 0.007, 0.997 ± 0.006, 1.003 ± 0.007, and 1.68 ± 0.07, resp. The transition state for the solvolysis reaction was modeled computationally using the exptl. KIE values as constraints. Taken together, the reported data are consistent with the retained solvolysis product being formed in an SNi (DN⧺*ANss) reaction with a late transition state in which cleavage of the glycosidic bond is coupled to the transfer of a proton from a solvating hexafluoro-2-propanol mol. In comparison, the inverted product, 1,6-anhydro-β-d-glucopyranose, is formed by intramol. capture of a solvent-equilibrated glucopyranosylium ion, which results from dissocn. of the solvent-sepd. ion pair formed in the rate-limiting ionization reaction (DN⧺ + AN). The implications that this model reaction have for the mode of action of retaining glycosyltransferases are discussed.
    37. 37
      Cowdrey, W. A.; Hughes, E. D.; Ingold, C. K.; Masterman, S.; Scott, A. D. Reaction kinetics and the Walden inversion Part VI Relation of steric orientation to mechanism in substitution involving halogen atoms and simple or substituted hydroxyl groups. Journal of the Chemical Society 1937, 0, 1252– 1271,  DOI: 10.1039/jr9370001252
      There is no corresponding record for this reference.
    38. 38
      Crich, D. Mechanism of a chemical glycosylation reaction. Acc. Chem. Res. 2010, 43 (8), 1144– 1153,  DOI: 10.1021/ar100035r
      38
      Mechanism of a Chemical Glycosylation Reaction
      Crich, David
      Accounts of Chemical Research (2010), 43 (8), 1144-1153CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Glycosylation is arguably the most important reaction in the field of glycochem., yet it involves one of the most empirically interpreted mechanisms in the science of org. chem. The β-mannopyranosides, long considered one of the more difficult classes of glycosidic bond to prep., were no exception to this rule. Several logical but circuitous routes for their prepn. were described in the literature, but they were accompanied by a greater no. of mostly ineffective recipes for their direct access. This situation changed in 1996 with the discovery of the 4,6-O-benzylidene acetal as a control element permitting direct entry into the β-mannopyranosides, typically with high yield and selectivity. The unexpected nature of this phenomenon demanded study of the mechanism, leading first to the demonstration of the α-mannopyranosyl triflates as reaction intermediates and then to the development of α-deuterium kinetic isotope effect methods to probe their transformation into the product glycosides. In this account, the authors assemble their observations into a comprehensive assessment consistent with a single mechanistic scheme. The realization that in the glucopyranose series the 4,6-O-benzylidene acetal is α- rather than β-directing led to further investigations of substituent effects on the stereoselectivity of these glycosylation reactions, culminating in their explanation in terms of the covalent α-glycosyl triflates acting as a reservoir for a series of transient contact and solvent-sepd. ion pairs. The function of the benzylidene acetal, as explained by Bols and co-workers, is to lock the C6-O6 bond antiperiplanar to the C5-O5 bond, thereby maximizing its electron-withdrawing effect, destabilizing the glycosyl oxocarbenium ion, and shifting the equil. as far as possible toward the covalent triflate. β-Selective reactions result from attack of the nucleophile on the transient contact ion pair in which the α-face of the oxocarbenium ion is shielded by the triflate counterion. The α-products arise from attack either on the solvent-sepd. ion pair or on a free oxocarbenium ion, according to the dictates of the anomeric effect. Changes in selectivity from varying stereochem. (glucose vs. mannose) or from using different protecting groups can be explained by the shifting position of the key equil. and, in particular, by the energy differences between the covalent triflate and the ion pairs. Of particular note is the importance of substituents at the 3-position of the donor; an explanation is proposed that invokes their evolving torsional interaction with the substituent at C2 as the chair form of the covalent triflate moves toward the half-chair of the oxocarbenium ion.
    39. 39
      Fu, Y.; Bernasconi, L.; Liu, P. Ab Initio Molecular Dynamics Simulations of the S(N)1/S(N)2 Mechanistic Continuum in Glycosylation Reactions. J. Am. Chem. Soc. 2021, 143 (3), 1577– 1589,  DOI: 10.1021/jacs.0c12096
      There is no corresponding record for this reference.
    40. 40
      Iglesias-Fernandez, J.; Hancock, S. M.; Lee, S. S.; Khan, M.; Kirkpatrick, J.; Oldham, N. J.; McAuley, K.; Fordham-Skelton, A.; Rovira, C.; Davis, B. G. A front-face ‘S(N)i synthase’ engineered from a retaining ‘double-S(N)2’ hydrolase. Nat. Chem. Biol. 2017, 13 (8), 874– 881,  DOI: 10.1038/nchembio.2394
      There is no corresponding record for this reference.
    41. 41
      Lee, S. S.; Hong, S. Y.; Errey, J. C.; Izumi, A.; Davies, G. J.; Davis, B. G. Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase. Nat. Chem. Biol. 2011, 7 (9), 631– 638,  DOI: 10.1038/nchembio.628
      41
      Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
      Lee, Seung Seo; Hong, Sung You; Errey, James C.; Izumi, Atsushi; Davies, Gideon J.; Davis, Benjamin G.
      Nature Chemical Biology (2011), 7 (9), 631-638CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
      A previously detd. crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group 18O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Bronsted anal. of nucleophiles and Taft anal. highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quant. data substantiate this unusual enzymic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.
    42. 42
      Paparella, A. S.; Cahill, S. M.; Aboulache, B. L.; Schramm, V. L. Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an S(N)i Mechanism and Ion Pair Transition State. ACS Chem. Biol. 2022, 17 (9), 2507– 2518,  DOI: 10.1021/acschembio.2c00408
      42
      Clostridioides difficile TcdB Toxin Glucosylates Rho GTPase by an SNi Mechanism and Ion Pair Transition State
      Paparella, Ashleigh S.; Cahill, Sean M.; Aboulache, Briana L.; Schramm, Vern L.
      ACS Chemical Biology (2022), 17 (9), 2507-2518CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      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 stereochem. 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 stereochem., while hydrolysis of UDP-glucose by TcdB causes inversion of stereochem. Kinetic anal. revealed TcdB glucosylation via the formation of a ternary complex with no intermediate, supporting an SNi mechanism with nucleophilic attack and leaving group departure occurring on the same face of the glucose ring. Kinetic isotope effects combined with quantum mech. calcns. revealed that the transition states of both glucohydrolase and glucosyltransferase activities of TcdB are highly dissociative. Specifically, the TcdB glucosyltransferase reaction proceeds via an SNi 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.
    43. 43
      Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 2008, 77 (0066–4154 (Print)), 521– 555. DOI: 10.1146/annurev.biochem.76.061005.092322
      43
      Glycosyltransferases: structures, functions, and mechanisms
      Lairson, L. L.; Henrissat, B.; Davies, G. J.; Withers, S. G.
      Annual Review of Biochemistry (2008), 77 (), 521-555CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
      A review. Glycosyltransferases catalyze glycosidic bond formation using sugar donors contg. a nucleoside phosphate or a lipid phosphate leaving group. Only 2 structural folds, GT-A and GT-B, have been identified for the nucleotide sugar-dependent enzymes, but other folds are now appearing for the sol. domains of lipid phosphosugar-dependent glycosyltransferases. Structural and kinetic studies have provided new insights. Inverting glycosyltransferases utilize a direct displacement SN2-like mechanism involving an enzymic base catalyst. Leaving group departure in GT-A fold enzymes is typically facilitated via a coordinated divalent cation, whereas GT-B fold enzymes instead use pos. charged side-chains and/or hydroxyls and helix dipoles. The mechanism of retaining glycosyltransferases is less clear. The expected 2-step double-displacement mechanism is rendered less likely by the lack of conserved architecture in the region where a catalytic nucleophile would be expected. A mechanism involving a short-lived oxocarbenium ion intermediate now seems the most likely, with the leaving phosphate serving as the base.
    44. 44
      Romero-Tellez, S.; Lluch, J. M.; Gonzalez-Lafont, A.; Masgrau, L. Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus beta-Glycosidase. A Combined MD and QM/MM Study. Front. Chem. 2019, 7, 200,  DOI: 10.3389/fchem.2019.00200
      There is no corresponding record for this reference.

      Original Research

    45. 45
      Bowles, W. H. D.; Gloster, T. M. Sialidase and Sialyltransferase Inhibitors: Targeting Pathogenicity and Disease. Front. Mol. Biosci. 2021, 8, 705133  DOI: 10.3389/fmolb.2021.705133
      45
      Sialidase and sialyltransferase inhibitors: targeting pathogenicity and disease
      Bowles, William H. D.; Gloster, Tracey M.
      Frontiers in Molecular Biosciences (2021), 8 (), 705133CODEN: FMBRBS; ISSN:2296-889X. (Frontiers Media S.A.)
      A review. Sialidases (SAs) and sialyltransferases (STs), the enzymes responsible for removing and adding sialic acid to other glycans, play essential roles in viruses, bacteria, parasites, and humans. Sialic acid is often the terminal sugar on glycans protruding from the cell surface in humans and is an important component for recognition and cell function. Pathogens have evolved to exploit this and use sialic acid to either "cloak" themselves, ensuring they remain undetected, or as a mechanism to enable release of virus progeny. The development of inhibitors against SAs and STs therefore provides the opportunity to target a range of diseases. Inhibitors targeting viral, bacterial, or parasitic enzymes can directly target their pathogenicity in humans. Excellent examples of this can be found with the anti-influenza drugs Zanamivir (Relenza, GlaxoSmithKline) and Oseltamivir (Tamiflu, Roche and Gilead), which have been used in the clinic for over two decades. However, the development of resistance against these drugs means there is an ongoing need for novel potent and specific inhibitors. Humans possess 20 STs and four SAs that play essential roles in cellular function, but have also been implicated in cancer progression, as glycans on many cancer cells are found to be hyper-sialylated. While much remains unknown about how STs function in relation to disease, it is clear that specific inhibitors of them can serve both as tools to gain a better understanding of their activity and form the basis for development of anti-cancer drugs. Here we review the recent developments in the design of SA and ST inhibitors against pathogens and humans.
    46. 46
      Mehr, K.; Withers, S. G. Mechanisms of the sialidase and trans-sialidase activities of bacterial sialyltransferases from glycosyltransferase family 80. Glycobiology 2016, 26 (4), 353– 359,  DOI: 10.1093/glycob/cwv105
      There is no corresponding record for this reference.
    47. 47
      Sugiarto, G.; Lau, K.; Qu, J.; Li, Y.; Lim, S.; Mu, S.; Ames, J. B.; Fisher, A. J.; Chen, X. A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating LewisX. ACS Chem. Biol. 2012, 7 (7), 1232– 1240,  DOI: 10.1021/cb300125k
      47
      A Sialyltransferase Mutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities for Directly Sialylating Lewisx
      Sugiarto, Go; Lau, Kam; Qu, Jingyao; Li, Yanhong; Lim, Sunghyuk; Mu, Shengmao; Ames, James B.; Fisher, Andrew J.; Chen, Xi
      ACS Chemical Biology (2012), 7 (7), 1232-1240CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Glycosyltransferases are important catalysts for enzymic and chemoenzymic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-contg. acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymic synthesis of complex sialyl Lewisx antigens contg. different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.
    48. 48
      Nin-Hill, A.; Rovira, C. The Catalytic Reaction Mechanism of the β-Galactocerebrosidase Enzyme Deficient in Krabbe Disease. ACS Catal. 2020, 10 (20), 12091– 12097,  DOI: 10.1021/acscatal.0c02609
      There is no corresponding record for this reference.
    49. 49
      Franconetti, A.; Arda, A.; Asensio, J. L.; Bleriot, Y.; Thibaudeau, S.; Jimenez-Barbero, J. Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions. Acc. Chem. Res. 2021, 54 (11), 2552– 2564,  DOI: 10.1021/acs.accounts.1c00021
      49
      Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions
      Franconetti, Antonio; Arda, Ana; Asensio, Juan luis; Bleriot, Yves; Thibaudeau, Sebastien; Jimenez-barbero, Jesus
      Accounts of Chemical Research (2021), 54 (11), 2552-2564CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)
      A review. Carbohydrates (glycans, saccharides, sugars) are essential mols. in all domains of life. Research on glycoscience spans from chem. to biomedicine, including material science and biotechnol. The access to pure and well-defined complex glycans using synthetic methods depends on the success of the employed glycosylation reaction. In most cases, the mechanism of the glycosylation reaction is supposed to involve the oxocarbenium ion. Understanding the structure, conformation, reactivity, and interactions of these glycosyl cations is essential to predict the outcome of the reaction. In this Account, building on our contributions on this topic, we discuss the theor. and exptl. approaches that have been employed to decipher the key features of glycosyl cations, from their structures to their interactions and reactivity. We also highlight that, from a chem. perspective, the glycosylation reaction can be described as a continuum, from unimol. SN1 with naked oxocarbenium cations as intermediates to bimol. SN2-type mechanisms, which involve the key role of counterions and donors. All these factors should be considered and are discussed herein. The importance of dissociative mechanisms (involving contact ion pairs, solvent-sepd. ion pairs, solvent-equilibrated ion pairs) with bimol. features in most reactions is also highlighted. The role of theor. calcns. to predict the conformation, dynamics and reactivity of the oxocarbenium ion is also discussed, highlighting the advances in this field that now allow the access to the conformational preferences of a variety of oxocarbenium ions and their reactivities under SN1-like conditions. Specifically, the ground-breaking use of superacids to generate these cations is emphasized, since it has permitted characterizing the structure and conformation of a variety of glycosyl oxocarbenium ions in superacid soln. by NMR spectroscopy. We also pay special attention to the reactivity of these glycosyl ions that depends on the conditions, including the counterions, the possible intra- or intermol. participation of functional groups that may stabilize the cation and the chem. nature of the acceptor, either weak or strong nucleophile. We discuss recent investigations from different exptl. perspectives, which identified the involved ionic intermediates, estg. their lifetimes and reactivities and studying their interactions with other mols. In this context, we also emphasize the relationship between the chem. methods that can be employed to modulate the sensitivity of glycosyl cations and the way in which glycosyl modifying enzymes (glycosyl hydrolases and transferases) build and cleave glycosidic linkages in nature. This comparison provides inspiration on the use of mols. that regulate the stability and reactivity of glycosyl cations.
    50. 50
      Darby, J. F.; Gilio, A. K.; Piniello, B.; Roth, C.; Blagova, E.; Hubbard, R. E.; Rovira, C.; Davies, G. J.; Wu, L. Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V. ACS Catal. 2020, 10 (15), 8590– 8596,  DOI: 10.1021/acscatal.0c02222
      50
      Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V
      Darby, John F.; Gilio, Amelia K.; Piniello, Beatriz; Roth, Christian; Blagova, Elena; Hubbard, Roderick E.; Rovira, Carme; Davies, Gideon J.; Wu, Liang
      ACS Catalysis (2020), 10 (15), 8590-8596CODEN: ACCACS; ISSN:2155-5435. (American Chemical Society)
      α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. The authors solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-assocd. N-glycosylation.
    51. 51
      Biarnes, X.; Ardevol, A.; Iglesias-Fernandez, J.; Planas, A.; Rovira, C. Catalytic itinerary in 1,3–1,4-beta-glucanase unraveled by QM/MM metadynamics. Charge is not yet fully developed at the oxocarbenium ion-like transition state. J. Am. Chem. Soc. 2011, 133 (50), 20301– 20309,  DOI: 10.1021/ja207113e
      51
      Catalytic Itinerary in 1,3-1,4-β-Glucanase Unraveled by QM/MM Metadynamics. Charge Is Not Yet Fully Developed at the Oxocarbenium Ion-like Transition State
      Biarnes, Xevi; Ardevol, Albert; Iglesias-Fernandez, Javier; Planas, Antoni; Rovira, Carme
      Journal of the American Chemical Society (2011), 133 (50), 20301-20309CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Retaining glycoside hydrolases (GHs), key enzymes in the metab. of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a 1,4B/1S3 conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction (1,4B/1S3 -4E/4H3 - 4C1) and shows that the transition state is not the point of max. charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.
    52. 52
      Davies, G. J.; Ducros, V. M.; Varrot, A.; Zechel, D. L. Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography. Biochem. Soc. Trans. 2003, 31 (Pt 3), 523– 527,  DOI: 10.1042/bst0310523
      There is no corresponding record for this reference.
    53. 53
      Stoddart, J. F. Stereochemistry of carbohydrates; Wiley-Interscience: New York, 1971.
      There is no corresponding record for this reference.
    54. 54
      Schuman, B.; Evans, S. V.; Fyles, T. M. Geometric attributes of retaining glycosyltransferase enzymes favor an orthogonal mechanism. PLoS One 2013, 8 (8), e71077  DOI: 10.1371/journal.pone.0071077
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

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    Accession Codes

    Atomic coordinates and structural factors for the reported 8 crystal structures have been deposited in the Protein Data Bank under the accession number (8ZWR, 8ZWP, 8ZWW, 8ZWY, 8ZX2, 8ZX3, 8ZX8, and 8ZX9). Other data are available from the corresponding author upon reasonable request.


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