Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

The soluble catalytic domain of human FUT8 was expressed as a secreted and N-terminally His-tagged GFP fusion protein in HEK293 cells as previously described for the production and biochemical characterization of numerous human glycosyltransferases (Fig. S1A) (). Purification of the enzyme from the conditioned media, fusion tag cleavage, and further purification led to an enzyme preparation amenable to enzymatic and structural studies (Fig. S1B). The oligomeric structure of FUT8 was examined by size exclusion-multiangle light scattering (SEC-MALS) revealing a single peak eluting with a predicted molecular mass of ∼132 kDa consistent with a predominately dimeric form for the ∼62.6 kDa expression product with tags removed (Fig. S1C).

Prior studies have focused on kinetic and inhibition studies using various donor substrates and analogs that profiled the contributions of the respective nucleoside, diphosphate, and sugar components (). Acceptor specificities have also been profiled using a collection of N-glycoprotein (, , ), glycopeptide (, , , , ), and glycan-Asn substrates (, , ), or glycan arrays (, ) of acceptors, but analysis of acceptor kinetics has been restricted to fluorophore-tagged or glycopeptide-linked A2-Asn. Here, we have employed a panel of Asn-linked glycan substrates to assess activity and kinetic constants using GDP-Glo assays. Of the various substrates, the A1-Asn and A2-Asn substrates were shown to display the optimal catalytic efficiencies (k_cat/Km) (Table S1 and Fig. 1), which is consistent with prior data demonstrating a requirement for the full GlcNAc-β1,2-Man-α1,3-Man- arm for high affinity substrate recognition (). The Km value for the A2-Asn substrate was higher (52 μm) than values previously reported for the fluorophore-tagged (12 μm ()) or glycopeptide-linked A2-Asn (13 μm ()) substrates consistent with the prior data indicating that extensions to the peptide backbone increases the affinity of the acceptor substrate (, ). The addition of a β1,4-GlcNAc residue to the GlcNAc-β1,2-Man-α1,3-Man arm (A3‘-Asn) reduced catalytic efficiency by 4.1-fold (Fig. 1). Similarly, extension of the GlcNAc-β1,2-Man-α1,6-Man arm with a β1,4-Gal residue (G1-Asn) reduced catalytic efficiency by 4.8-fold, whereas additional branching to form A3-Asn or incomplete mannose trimming to form NM5N2-Asn reduced catalytic efficiency by 19.8- and 8.3-fold, respectively (Fig. 1). Substrates lacking the β1,2-GlcNAc on the Man-α1,3-Man arm (M5N2-Asn and M3N2-Asn intermediates) had drastically reduced catalytic efficiencies (1590-fold reduction and no detectable activity, respectively). Finally, a tetra-antennary substrate (A4-Asn) had no detectable activity as acceptor. These data, in combination with previous results on relative acceptor specificities (, , , , , ), indicate that the unmodified GlcNAc-β1,2-Man-α1,3-Man arm in both A1-Asn and A2-Asn are the critical determinants for high affinity substrate recognition, and additional β1,4-GlcNAc branching (A3‘-Asn) on the subterminal Man residues or modifications on the GlcNAc-β1,2-Man-α1,6-Man arm (A3-Asn, A4-Asn, NM5N2-Asn, or G1-Asn) interferes with acceptor recognition (Fig. 1). Thus, FUT8 acceptor specificity exhibits a stringent exclusion of substrates containing terminal glycan branching beyond the A2-Asn core structure.

Structural studies on FUT8 followed similar approaches that have been taken with other recent glycosyltransferase structures (, , , ) and other recent structures of human FUT8:A2-Asn complexes (, ). The soluble catalytic domain of FUT8 was initially crystallized in the presence of 5 mm GDP. X-ray diffraction data were collected to 2.25 Å resolution and the structure was solved by molecular replacement using the prior apo FUT8 structure (PDB 2DE0 ()) as a search model. There were four chains in the asymmetric unit of the FUT8:GDP complex. The overall structure was comprised of a GT-B–fold but, as noted in the prior FUT8 structures (, , ), the enzyme has three large helical segments N-terminal to the GT-B–fold (two involved in homodimerization, see below) and an SH3 domain near the C terminus (Fig. 2).

The SEC-MALS data for the purified enzyme indicated that FUT8 is a dimer in solution (Fig. S1). Similar to recent analysis of FUT8 structures (, ), the asymmetric unit of the present FUT8:GDP complex shows that chains B and C form a dimer with an interface that buries 3192 Ų of surface area (Fig. 2). Chains A and D form analogous dimers with their crystallographically related symmetry mates. PISA analysis indicates that the interface is stable, with a favorable solvation free-energy gain (ΔⁱG) of −29.8 kcal mol⁻¹ and a p value of 0.057, which corresponds to a high likelihood that the surface is interaction-specific () The dimer interface is formed from the first two N-terminal α-helices of FUT8, which fold into a coiled-coil helix structure that associates with the corresponding coiled-coil pairs from adjacent subunits to form a 4-helix bundle, which buries heptad repeat residues in the hydrophobic core (Fig. 2, A and B) (). Although this 4-helix bundle interaction was recognized in the recent FUT8:GDP:A2-Asn structures (, , ), it is notable that this dimer interface is also stabilized by extensive H-bonding and van der Waals interactions between the exposed face of the helical hairpin from one chain and the SH3 domain and residues from the GT-B–fold (residues 388-396) from the adjacent chain (Fig. 2A). Interestingly, the interactions between the SH3 domain and the lateral face of the 4-helix do not involve prolines, despite the fact the interface is formed from same loop regions that canonical SH3 domains use to recognize proline-rich ligand sequences () (Fig. 2A). The N termini of the two chains are adjacent at the base of the 4-helix bundle, but ∼77 amino acids of N-terminal “stem region” () were not resolved in the present structure and would be linked to the type 2 transmembrane segment in the full-length FUT8 enzyme (Fig. 2C). The program DISOPRED3 () predicts that the linker region of human FUT8 will most likely be intrinsically disordered.

Although the overall fold of the FUT8:GDP complex was similar to the prior apo structure (PDB 2DE0 ()) and the recent FUT8:GDP:A2 complexes (, ), there were notable differences in conformations of several loops between each of the four chains in the GDP complex and also with the previously published structures. The loop regions are comprised of Loop 1 (residues 245-273), Loop 2 (residues 365-378), and Loop 3 (residues 436-443) (Fig. 3B). These three flexible loops enclose the GDP-binding subsite. Chains A and B of the FUT8:GDP complex contained unambiguous electron density for a well-ordered GDP, chain C contains a weakly ordered GDP, and chain D contains no density for the nucleotide (Fig. 3, A and B). The conformations and ordering of the three loops are strongly correlated with the occupancy of bound ligand. The fact that the crystals were grown in saturating GDP suggests that variable occupancy of the nucleotide is due to the crystal lattice selecting for loop conformations that weakened binding of the donor. Thus, in solution it is likely that these loops are flexible and undergo an induced fit folding upon binding of the donor substrate (Fig. 3D). The structures of each of the chains will be discussed separately below.

Chains A and B of the FUT8:GDP complex exhibit full occupancy for the bound GDP and Loops 1, 2, and 3 interact with the bound nucleotide (Fig. 3, A and B). The loop conformations of chain A are in analogous positions to the previously described FUT8:GDP:A2-Asn complexes (, ). Loop 2 harbors Arg³⁶⁵ and Lys³⁶⁹, which have direct ionic interactions with the β-phosphate oxygen of the nucleotide and help to enclose the bound nucleotide (Fig. 4, A and B). Minor differences in Loop 2 conformations are seen between chains A and B and are likely due to crystal packing (Fig. 3C). Loop 3 adopts a single conformation in chains A and B, and contributes significant packing interactions to the nucleotide and ribose through Ala⁴³⁶ and Arg⁴⁴¹, respectively. In addition, Arg⁴⁴¹ forms a complex ion pairing interaction with Asp³⁶⁸ in Loop 2, which in turn bridges to Arg³⁶⁵ in Loop 2 to completely enclose the bound nucleotide (the “Arg³⁶⁵-Asp³⁶⁸-Arg⁴⁴¹ cage” (Fig. 4C). Finally, Tyr²⁵⁰ in Loop 1 forms an H-bond with the O2 ribose hydroxyl of the bound GDP in both chains A and B (Fig. 5, A and B). The β-phosphate has additional interactions with the side chain hydroxyl of Ser⁴⁶⁹ and the backbone nitrogen of Gln⁴⁷⁰, whereas the α-phosphate has polar interactions with the backbone nitrogen atoms of Gly²²¹ and Cys²²². The extensive electrostatic interactions with the β-phosphate likely explains why GDP is a much more potent inhibitor of FUT8 (3.6 μm Ki) than GMP (2.3 mm Ki) (). The ribose O3 hydroxyl also H-bonds with the peptide nitrogen of Tyr²²⁰, and the purine ring of the nucleoside interacts with His³⁶³, Thr⁴⁰⁸, and Asp⁴⁵³ (Fig. 4, A and B). There are additional minor differences in conformation for solvent-exposed side chains between chains A and B, but interactions with the bound GDP are conserved.

In contrast, chain C has significantly weaker electron density for the bound GDP (Fig. 3, A and B) and a significant portion of Loops 2 and 3 are completely disordered (indicated by red and blue spheres in Fig. 3B, upper panels). Only residues 365-366 and 375-376 are ordered in Loop 2 and residues 430-432 are ordered in Loop 3. The conformation of Loop 1 in chain C is similar to chains A and B with Tyr²⁵⁰ hydrogen bonding with the ribose O2 hydroxyl of the bound GDP. The disorder of Loops 2 and 3 opens the Arg³⁶⁵-Asp³⁶⁸-Arg⁴⁴¹ cage and exposes the GDP to solvent (Fig. 3B). The rest of the binding site retains the other H-bonding and ionic interactions between the GDP and the polypeptide similar to chains A and B. Thus, the loss of the Arg³⁶⁵-Asp³⁶⁸-Arg⁴⁴¹ cage and the associated electrostatic and packing interactions likely explains the lower occupancy of the nucleotide.

Chain D has no visible density for GDP in the binding site (Fig. 3, A and B). The disorder for Loop 2 was similar to chain C, but residues 430-435 in Loop 3 are positioned to indicate that this peptide segment is extended away from the GDP-binding site reminiscent of the position of the same loop in the PDB 2DE0 structure. A similar loss of binding residues is found for Loops 2 and 3 compared with chain C. In addition, Loop 1 in chain D is altered and does not position Tyr²⁵⁰ within H-bonding distance of the ribose O₂ hydroxyl in the nucleotide. Thus, loss of interactions with all three loops explains the absence of a bound GDP in chain D.

Finally, a comparison with PDB 2DE0 shows that the loops in the unliganded structure are similar in flexibility to those in chain D (Fig. 3B). In PDB 2DE0, Loop 2 extends away from the active site with residues 373-376 being disordered. Although Loop 3 is ordered in PDB 2DE0, it has moved >25 Å away from the GDP-binding site. The conformation of Loop 1 is similar to that observed in the FUT8:GDP chain D. These altered conformations are additional evidence that all three loops are likely disordered in solution, and supports an induced-fit folding upon nucleotide binding (Fig. 3D).

To examine the acceptor substrate interactions, we crystallized a FUT8:GDP:A2-Asn complex. The structure was solved at 2.4 Å resolution in space group P6₅22 with two chains in the asymmetric unit. Application of crystallographic symmetry reveals the same dimer that we previously observed. The two chains are essentially the same (rmsd 0.20 Å for 464 Cα atoms) with the exception of minor surface loops and solvent-exposed side chain differences. The structure is similar to chain A of the FUT8:GDP (rmsd 0.34 Å for 459 Cα atoms), with the exception that the conformation of Loop 2 is significantly different from the rest of the chains in the FUT8:GDP complex.

Both chains in the FUT8:GDP:A2-Asn complex display well-defined electron density for GDP and the entire A2-Asn molecule, which binds in a complementary surface groove on the face of the GT-B–fold and extends from a position adjacent to the GDP-binding site toward the SH3 domain (Fig. 5A). The FUT8:GDP:A2-Asn structure and its ligand interactions (Fig. 5, B and C) are essentially identical to those identified in the recently published FUT8:GDP:A2 complexes (, ) (the rmsd range from 0.45 to 0.54 Å for 450-467 corresponding Cα atoms in PDB 6TKV ()). Briefly, GlcNAc residue N1 (Fig. 5) is positioned adjacent to the GDP-binding site with the O6 hydroxyl facing toward the GDP ligand through H-bonding interactions with Glu³⁷³. This residue is positioned in Loop 2 and is fully ordered in its interaction with the hydroxyl nucleophile of the acceptor. Glu³⁷³ also interacts through a salt bridge to Lys³⁶⁹ and subsequently to the β-phosphate oxygen of the GDP. These interactions suggest that Glu³⁷³ acts as the catalytic base to deprotonate the N1 O6 nucleophilic hydroxyl and then in turn protonate the phosphate of the leaving GDP product (see “Catalytic Mechanism” below). Additional interactions include H-bonds from the N1 O3 hydroxyl to Asp²⁹⁵ and Lys²¹⁶, the Asn amide nitrogen with Gly²¹⁷, and hydrogen bonds between Gln⁴⁷⁰ and the N2 N-acetyl glycosidic oxygen. A further matrix of hydrogen bonding interactions extends throughout the length of the A2-Asn structure all the way to the nonreducing terminal GlcNAc residues (N3 and N4). Many of the interactions with the terminal A2-Asn residues are contributed by the SH3 domain (Fig. 5B, green residue labeling with green boxes). All of the monosaccharides in the acceptor have at least one H-bond to the enzyme surface with the exception of the internal Man residue in the GlcNAc-β1,2-Man-α1,3-Man arm (M2 residue). The details of the acceptor interactions are shown in Fig. 5, B and C. Most notable among these interactions is the position of the SH3 domain, which sterically blocks the linear extension of the A2 glycan beyond the β-linked Man residue (M1) and leads to a bifurcation of the two terminal glycan branches in opposite directions across the surface of the SH3 domain. Each branch has interactions with the SH3 domain including residues N3, M3, and N4. The close interaction of the M1 residue with the SH3 domain surface also precludes binding of glycans containing a bisecting GlcNAc residue.

To confirm the contributions of binding site amino acids to acceptor glycan interactions we mutated each of the respective amino acids to alanine, tested the activity, and performed kinetic analysis on the active mutants (Fig. 6C). A subset of the residues in the GDP-binding site had previously been tested for enzyme activity () and we confirmed that mutation of these and other residues that directly interact with the GDP lead to complete enzyme inactivation (Table S3 and Fig. 6, B and C). In addition, we probed residues that contribute to acceptor glycan interactions and found that mutation of three residues, Asp²⁹⁵ that H-bonds with the O3 hydroxyl of N1, Tyr⁴⁹⁸ that provides van der Waals packing interactions with the hydrophobic face of the β-Man residue (M1), and Asp⁴⁹⁵ that H-bonds with M1 and the terminal N3 residue, all lead to complete loss of enzyme activity (Fig. 6A). Mutations of other interacting residues result in significantly decreased catalytic efficiency, but not complete inactivation (Fig. 6C). These reductions in k_cat/Km include Lys²¹⁶ that H-bonds with N1 (160-fold reduction in k_cat/Km), Gln⁴⁷⁰ that H-bonds with N2 (80-fold reduction), Asp⁴⁹⁴ adjacent to N3 (800-fold reduction), His⁵³⁵ that H-bonds with N3 (11-fold reduction), and Gln⁵⁰² that H-bonds with N4 (16-fold reduction). A similar reduction in activity was previously seen for mutants in the SH3 domain including H535A as well as H535A/Lys541A and T550A/L552A double mutants in prior studies () and a collection of residues conserved between human FUT8 and chicken c-Src () demonstrating the critical role of the SH3 domain in providing acceptor substrate specificity and affinity. Thus, whereas the donor-binding site is exceptionally sensitive to mutational perturbation, the acceptor-binding site has fixed points of essential interaction and other regions that contribute incremental binding energy to catalytic efficiency.

The kinetic studies with varied acceptor substrate structures indicated a preference for glycans containing an unmodified GlcNAc-β1,2-Man-α1,3-Man arm (A1-Asn and A2-Asn structures), and reduced activities for substrates that harbor additional modifications to the core A2-Asn structure. The impacts of these modifications on k_cat/Km are highlighted in Fig. 7A. To examine substrate interactions with alternative acceptor structures we performed additional co-crystallization studies with A3'-Asn (Fig. 7B), A3-Asn (Fig. 7C), and NM5N2-Asn (Fig. 7D) acceptors. In each case we obtained well-defined electron density for at least a portion of the corresponding acceptor glycan.

The structure of the FUT8:GDP:A3'-Asn complex was solved at 3.3 Å resolution, in the same space group as FUT8:GDP:A2-Asn. FUT8:GDP:A3'-Asn has two chains in the asymmetric unit and both chains display unambiguous electron density for the GDP and for all residues of the A3'-Asn acceptor. The overall enzyme structure was similar to the two chains of the FUT8:GDP:A2-Asn complex (rmsd 0.22-0.29 Å for 465 Cα atoms between the corresponding chains) and the donor and acceptor interactions within the respective binding subsites were identical to the FUT8:GDP:A2-Asn complex. The major difference was the presence of an extra β1,4-GlcNAc residue (N5) on the GlcNAc-β1,2-Man-α1,3-Man arm that distinguishes the A3'-Asn structure from A2-Asn (Fig. 7B and inset). In both chains, the N5 residue faces into the solvent and does not contribute directly to acceptor-binding interactions.

The structure of the FUT8:GDP:A3-Asn complex was determined at 2.47 Å resolution in space group P6₅ with eight chains in the asymmetric unit. All eight chains display unambiguous electron density for the GDP, but density for the acceptor was evident only in chains A, D, E, and H. In each case where the acceptor density was present, only a single distal arm and the core of the glycan structure (GlcNAc-β1,2-Man-α1,3-Man-β1,4-GlcNAc-β1,4-GlcNAc-β-Asn) was resolved (Fig. 7C). The Man-α1,6- (M3) branching residue from the core β-Man (M1) was disordered along with the two distal GlcNAc residues (N4 and N6) extending from this arm (Fig. 7C and inset). The disorder for these three monosaccharides in the acceptor complex indicates that the predicted steric hindrance (Fig. 7A) for the additional β1,6-GlcNAc residue (N6) altered the positioning of the entire branch.

The structure of the FUT8:GDP:NM5N2-Asn complex was determined at 3.2 Å resolution. Similar to FUT8:GDP:A3-Asn, this structure was also in space group P6₅ with eight chains in the asymmetric unit. For this complex, the electron density for both the GDP and the GlcNAc-β1,2-Man-α1,3-[Man-α1,6-]Man-β1,4-GlcNAc-β1,4-GlcNAc-Asn portion of the acceptor was visible in all eight chains, although the order of the GlcNAc-Asn region of the acceptor varied between chains (Fig. 7D and inset). The electron density for the M3 branch extending from the core β-Man (M1) is well-defined. However, density for the M4 and M5 residues was weak and they were left unmodeled.

The steric constraints of the acceptor-binding site clearly indicate that the 1,6-substituents on the M3 residue in the A3-Asn and NM5N2:Asn complexes are not compatible with the same binding geometry as the A2-Asn structure (GlcNAc-β1,6- (N6) for A3-Asn and Man-α1,6- (M5) for NM5N2-Asn). These modifications to the M3 residue lead to rotation or disorder of the more flexible Man-α1,6- linkage between M3 and M1 to accommodate the bulky branched substituents and likely account for the reduced catalytic efficiencies for these glycan acceptors.

Previous sequence (), HCA (), and structural () alignments of fucosyltransferases have compared α-1,2- and α-1,6-fucosyltransferases with mammalian protein-O-fucosyltransferases (POFUTs) and identified three conserved sequence motifs that are common among the sequence families (Fig. S2, red boxes), including residues involved in binding the nucleotide portion of the sugar donor (). Structures are now available for mouse (), human (), and Caenorhabditis elegans () POFUT1 (GT), C. elegans (), and human () POFUT2 (GT68), Arabidopsis FUT1 (GT37 (, )), NodZ (GT23, (, )), and human FUT8 (GT28) and structural alignment using the PROMALS3D server () allowed us to make a more comprehensive comparison between these enzymes to identify additional conserved structural elements. The resulting alignment revealed extensive structural similarity far beyond the three previously identified motifs (Fig. S2, blue boxes). Visualization of these conserved elements (Fig. S3A, Conserved) indicated the core of both Rossmann-folds were highly conserved among these enzymes including more than eight helical segments and four of the β-strands within the Rossmann-fold associated with the donor-binding site. Six β-strands of the second Rossmann-fold are less conserved in position, but still maintain a high degree of structural similarity among the collection of enzymes (Fig. S3, A and B). The degree of structural similarity is striking considering pairwise primary sequence similarities were <15%. The PROMALS3D alignment also identified 13 amino acids that are most conserved among the structures, some of which were noted in a more focused comparative analysis of the sugar donor-binding site (). Displaying these amino acids within the framework of the FUT8 structure shows that 9 of the 13 amino acids are clustered within the base of the GDP-binding site proximal to the Rossmann-fold (Fig. S3C) and either directly interact with the donor or contribute to the architecture of the donor site. The other four residues are distributed more widely within the FUT8 structure and do not interact with substrates or contribute to catalysis. For FUT8, the conserved binding site residues are not a part of Loops 1-3 that undergo conformational changes upon donor binding and, surprisingly, these three dynamic loops are not conserved in position or sequence among the other fucosyltransferase structures.

In contrast to the similarity in the underlying Rossmann-fold and donor-binding subsite, each of the enzymes are entirely different in their modes of interactions with their respective acceptor molecules (Fig. S3). Although the positions of acceptor interactions are approximately similar in the clefts between the two Rossmann-folds, each enzyme employs unique loop insertions into the Rossmann-fold scaffold to assemble the acceptor binding subsites (Fig. S3). For the POFUTs that recognize larger protein domain structures, the interactions are assembled through the generation of a deep cleft between the two subdomains. For the glycan-directed fucosyltransferases (FUT8 and AtFUT1), the interactions are through a more flattened face of the cleft and, for FUT8, the recruitment of an additional SH3 domain insertion near the C terminus. This clearly indicates the presence of evolutionary divergence in the respective loop regions to provide unique acceptor binding specificities, whereas the core scaffold of the GT-B structure and donor interactions are more conserved.

An even more striking observation from the aligned structures is the similarities in positions for the acceptor nucleophiles in the respective structures (Fig. 8B). FUT8 and AtFUT1 (, ) structures have been solved as extended glycan acceptor complexes, whereas POFUT1 () and POFUT2 () have been solved in complex with EGF and thrombospondin repeat domain acceptors, respectively. In each instance, the corresponding acceptor hydroxyl has been identified in a position for nucleophilic attack. Surprisingly, despite the differences in the respective acceptor structures, the nucleophilic hydroxyls are positioned similarly. The nucleophilic hydroxyls for the two glycan-directed enzymes (FUT8 and AtFUT1) superimpose almost exactly. Similarly, the nucleophilic Ser/Thr hydroxyls for the POFUTs also superimpose with each other at a position <2 Å from the nucleophiles for the glycan-directed enzymes.

Among the six aligned fucosyltransferases in Fig. S3, only two have a clearly identified catalytic base (FUT8 and POFUT2 ()). POFUT1 and AtFUT1 have no apparent ionizable side chain in proximity to the acceptor hydroxyl and are proposed to use a S_N1 mechanism employing a proton shuttle (, , , ). NodZ was not solved as an acceptor complex and its catalytic base was not identified (, ).

To identify the catalytic mechanism of FUT8 we superimposed the aligned structure of the intact GDP-Fuc from the POFUT2:GDP-Fuc complex () in the donor-binding site (Fig. 8A). This resulted in the placement of the Fuc residue in the FUT8 active site without steric hindrance and predicted H-bonds for the fucose O2 with the backbone nitrogen of Gly²¹⁹ and for O4 with Asp³⁶⁸ (from Loop 2). This latter interaction is consistent with the loss of FUT8 activity for the D368A mutant (Table S3). In addition, C1 of the Fuc residue is directly in line for nucleophilic attack by the O6 hydroxyl of N1 (Fig. 8A). Thus, the structure of the FUT8:GDP:A2-Asn complex and the modeled donor confirm the inverting catalytic mechanism of the base-catalyzed S_N2 nucleophilic attack of the sugar nucleotide donor by the N1 O6 hydroxyl.

Although both FUT8 and POFUT2 perform similar S_N2 in-line nucleophilic attacks of their respective GDP-Fuc donors, the catalytic base for POFUT2 (E52 ()) originates from a completely different region of the protein structure compared with FUT8 (Fig. 8B). The catalytic base (Glu³⁷³) for FUT8 is on the opposite side of its acceptor nucleophile relative to Glu⁵² in POFUT2, whereas POFUT1 and AtFUT1 do not have ionizable side chains at either position. Clearly, whereas the enzymes conserve residues for donor and nucleophile binding, the constraints for positioning the catalytic base and enzymatic mechanism are not conserved among these enzymes.