Abstract
Super-armed glycosyl donors, whose substituents are predominantly held in pseudoaxial positions, exhibit strongly increased reactivity in glycosylation through significant stabilization of oxocarbenium-like transition states. Examination of X-ray crystal structures reveals that the GH47 family of glycoside hydrolases have evolved so as to distort their substrates away from the ground state conformation in such a manner as to present multiple C-O bonds in pseudoaxial positions and so benefit from conformational super-arming of their substrates, thereby enhancing catalysis. Through analysis of literature mutagenic studies, we show that a suitably placed aromatic residue in GHs 6 and 47 sterically enforces super-armed conformations on their substrates. GH families 45, 81, and 134 on the other hand impose conformational super-arming on their substrates, by maintaining the more active ring conformation through hydrogen bonding rather than steric interactions. The recognition of substrate super-arming by select GH families provides a further parallel with synthetic carbohydrate chemistry and nature and opens further avenues for the design of improved glycosidase inhibitors.
Keywords: Glycoside hydrolases, conformational analysis, super-arming, α-mannosidases, twist-boat, half-chair, CH-π interactions, electrostatic transition state stabilization
Graphical Abstract
Introduction
Glycoside hydrolases (GHs) are carbohydrate processing enzymes that catalyze the cleavage of glycosidic bonds from either the terminal (exo-GHs) or internal (endo-GHs) positions of saccharides and oligosaccharides. They are found in all kingdoms of life and their absence or malfunction is associated with multiple human disease states, resulting in an immense interest in their mechanisms of action1–5 and in the design of inhibitors, ideally with selectivity for one class over another so as to limit toxicity.6–8 Multiple families (~170) of GH exist that, with certain exceptions (GH families 4, 88, 105, and 109),9–17 proceed by cleavage of the exocyclic glycosidic bond through what are considered to be exploded transition states that involve substantial oxocarbenium ion character, with either inversion or retention (double inversion) of configuration (Figure 1a–c) and the potential for substantial variation in the degree of synchronicity. The mechanisms of chemical glycosidic bond formation and hydrolysis resemble those of GH glycosidic bond hydrolysis in that they also typically proceed with substantial oxocarbenium ion character at the transition state,18–20 and as such it is not surprising that GHs and glycosyltransferases (GTs) have provided the inspiration for the development of new glycosylation reactions.21, 22
Figure 1.
(a) Mechanism of inverting glycoside hydrolases (b) Mechanism of retaining glycoside hydrolases (c) Concerted oxocarbenium-like transition state for an inverting glycosidase
In chemical glycosylation it has been established that of the three staggered conformations of the side chain (the exocyclic C5-C6 bond in the hexopyranoses and the C6-C7-bond in the ulosonic and neuraminic acids), the trans,gauche (tg) conformation destabilizes oxocarbenium-like transition states and so retards reactions, whereas the gauche,gauche (gg) conformation stabilizes oxocarbenium-like transition states and correspondingly accelerates reactions: the gauche,trans (gt) conformation displays intermediate behavior.23 This influence of exocyclic bond conformation on reactivity at the anomeric center arises from the relationship of the C-O bond in the side chain with the nascent positive charge at the transition state, either providing electrostatic stabilization (gg, and gt) or causing destabilization because of its electron-withdrawing nature (tg) (Figure 2 a–c).24–30 In keeping with the well-established principles of reactivity and the mechanistic parallels between chemical and enzymatic cleavage and formation of C-O bonds at the anomeric center, we recently demonstrated by analysis of available crystallographic data that GHs also employ restriction of side chain conformation to assist catalysis. Thus, an exhaustive search of the PDB with the aid of the Carbohydrate-Active Enzymes Database (CAZy, http://www.cazy.org)31 revealed that 84% of β-glucosidases, 75% of β-glucosaminidases, 99% of α-glucosidases, and 100% of α-glucosaminidases and β-mannosidases bind their substrates, substrate analogs, or transition state analog inhibitors1 with their side chains in the reactivity-enhancing gg conformation.32 These strikingly high populations of the gg conformation in molecules bound at the active site (−1 site)33 very significantly exceed those found in free solution (Figure 2a) and in complexes with lectin carbohydrate binding domains. In contrast, galactosidases bind their substrates with a class dependent preference for the gt or tg conformations, no doubt because of the relatively higher energy of the gg conformation in galactosides as reflected in the typical solution phase population distribution (Figure 2b).
Figure 2.
The three staggered side chain conformations and their approximate populations in free solution for (a) gluco- and mannopyranoses, and (b) for galactopyranoses. (c) Spatial relationships of side chain hydroxyl groups with the putative oxocarbenium π* orbital
The α-mannosidases (found in GHs 31, 38, 47, 63, 76, 92, 99, and 125),31 however, were found to constitute a broad exception to the very high preference for binding of the gg conformation exhibited by the α- and β-glucosidases and the β-mannosidases.32 Thus, of the 54 complexes located in the PDB meeting the criteria of ≤ 2.00 Å resolution and conformational integrity as judged by Privateer,34 25 had the side chain restricted by hydrogen-bonding to the gg and 28 to the gt conformations (with the remaining structure taking up an eclipsed conformation), at first sight suggesting that the α-mannosidases do not control side chain conformation to facilitate hydrolysis. Closer examination however revealed that, in point of fact, the 28 structures with the gt conformation were limited to only two GH families: 18 from GH38 and 10 from GH47, with all other α-mannosidases following the more typical behavior. Further, among all 170 GH families, only α-mannosidases from these two families together with those from GH92 (where the side chain is bound in the gg conformation) employ an essential divalent metal cation that chelates O2 and O3 of the ligand. We sought to understand these family specific exceptions to the broad general rule and present here our findings that Nature has again preempted chemists in the discovery and application of a principle of chemical reactivity, the conformational super-arming principle6, 35–42 for the stabilization of positive charge in glycosyl oxocarbenium-like transition states, and its application to the catalysis of glycosidic bond cleavage.
Results and Discussion
We first address the GH class 38 and 92 α-mannosidases for which Scheme 1a shows the conformational itinerary through the course of the hydrolysis reaction.43–47 The former use a retaining mechanism, with an active site aspartate as nucleophile, whereas the latter employ an inverting mechanism with water as a nucleophile. In each, the substrate binds in a OS2 twist boat conformation, passes through an approximate B2,5 boat-like transition state, and ends in a 1S5 twist boat as either a covalent glycosyl enzyme intermediate (GH38) or as the hydrolyzed product (GH92).48
Scheme 1.
(a) Conformational itinerary of pyranosides at the −1 site of GH38 and GH92 mannosidases with partial crystal structures of (b) D. melanogaster GH38 Golgi α-1,2-mannosidase bound to a mannoimidazole TS analog inhibitor (PDB ID 3D4Y), (c) D. melanogaster GH38 Golgi α-1,2-mannosidase bound to noeuromycin (PDB ID 2ALW), (d) B. thetaiotaomicron 3990 GH92 α-mannosidase bound to a mannoimidazole TS analog inhibitor (PDB ID 2WZS), and (e) B. thetaiotaomicron 3990 GH92 α-mannosidase bound to kifunensine (PDB ID 2WVZ). Blue dashed lines designate hydrogen bonds and purple dashed lines designate coordinative bonds to the metal.
GH38 mannosidases only bind Zn2+ in their active sites. Chelation of O2 and O3 by the metal facilitates twisting of the starting material from the ground state 4C1 chair to a skew boat closer in character to the B2,5 boat-like transition state, as suggested by the Rose and Bols groups in their analysis of binding of the inhibitor noeuromycin.45 Further, acidification of hydroxyl groups via coordination by Zn2+ is well established,49 with an observed reduction of the pKa of coordinated water to 9 in free solution and to 6 in carboxypeptidase active sites.50 Such acidification of O2 and O3 through chelation to Zn2+ affords significant transition state stabilization, as these groups are rendered less electron-withdrawing. In the GH38 mannosidases Zn2+ also coordinates to the aspartate nucleophile (Figure 3a)45 presumably orienting it for nucleophilic attack. Apparently, TS stabilization arising from chelation to Zn2+ in GH38s is such that no significant benefit is derived from enforcement of the gg side chain conformation.
Figure 3.
Coordination spheres of the divalent cation of (a) D. melanogaster GH38 golgi α-mannosidase II in complex with a mannoimidazole (Zn2+, PDB ID 2ALW), (b) B. thetaiotaomicron GH92 α-1,2-mannosidase in complex with a mannoimidazole (Ca2+, PDB ID 6F92), and (c) human GH47 α-1,2-mannosidase in complex with 1-deoxynojirimycin (Ca2+, PDB ID 1FO2). Blue dashed lines designate hydrogen bonds and purple dashed lines designate coordinative bonds to the metal.
In contrast, chelation to Ca2+ is significantly less activating as is apparent from the smaller reduction in the pKa of coordinated water to only 12.6.51, 52 While this does not preclude orientation of catalytic aspartate or glutamate residues, as observed with GH97 glucosidases53 and with B. thetaiotaomicron GH92 mannosidases (Figure 3b),54 Ca2+ does not significantly acidify O2 and O3 of the substrate. Rather, the Ca2+ serves principally to bind the ligand, as it is known to do in multiple lectins,55 and to orient the nucleophilic water.44 Consistent with the minimal activation provided by chelation to Ca2+, the Ca2+-dependent GH92 series of mannosidases impose the gg conformation of the side chain on their ligands so as to provide additional stabilization to the TS. In contrast to the GH92s, the Ca2+ dependent GH47 mannosidases benefit neither from metal-mediated activation of the catalytic aspartate (Figure 3c)56 nor from enforcement of the gg conformation.
The key to the high reactivity of the GH47 mannosidases is revealed by Davies’ study of the conformational itinerary followed by their substrates during hydrolysis (Scheme 2a).57 The substrate binds in a distorted 3S1 twist boat conformation, where, in contrast with GHs 38 and 92, the substituents at C3, C4, and C5 are all pseudoaxial, as observed with a non-hydrolyzable thioglycoside bound to Caulobacter sp. K31 (Scheme 2b). The pseudoaxial orientation of these C-O bonds is maintained over the course of the reaction as the substrate passes through a 3H4 half chair-like transition state, as seen with the complex of a mannoimidazole bound to the same enzyme (Scheme 2c), to the hydrolyzed product that is held as an “inverted” 1C4 chair conformation as exhibited by the Caulobacter sp. K31 complex with noeuromycin (Scheme 2d).
Scheme 2.
(a) Conformational itinerary of pyranosides at the −1 site of GH47 mannosidases, illustrated with partial crystal structures of Caulobacter sp. K31 in complex with (b) a thioglycoside (PDB ID 4AYP), (c) a mannoimidazole (PDB ID 4AYQ) 5KK7, and (d) noeuromycin (PDB ID 4AYR). Blue dashed lines designate hydrogen bonds and purple dashed lines designate coordinative bonds to the metal.
Further analysis of crystal structures of GH47 mannosidases reveals that a suitably placed aromatic residue (Figure 4)58 abuts the C4-C5-C6 plane of the bound pyranoside. As previously noted for arenes in the −1 site of the majority of GHs,59 this arene doubtless stabilizes the Michaelis complex through CH-π interactions,60 which are accentuated for H5 at the TS due to its proximity to the partial positive charge. We suggest, however, that a major function of this aryl group in proximity to C5 is to enforce distortion of the pyranoside ring at the −1 site away from the 4C1 chair so as to avoid a steric clash with the hydroxymethyl side chain. This is illustrated by mutagenic studies carried out by the Moremen group,58 where the F659A mutation in human ER α-mannosidase results in a 140-fold drop in kcat/KM indicating that removal of the steric bulk offered by F659 allows the substrate to take a more relaxed 4C1 or analogous conformation in the −1 site. Likewise, the F659A mutation results in a 60-fold drop in binding affinity for kifunensine, an inhibitor, whose ground state conformation is an inverted 1C4 chair, that binds near-irreversibly to the wild type enzyme.
Figure 4.
Partial crystal structure of human GH47 α-1,2-mannosidase bound to thiomannobiose, with F659 abutting the C4-C5-C6 plane (PDB ID 1X9D). Blue dashed lines designate hydrogen bonds and purple dashed lines designate coordinative bonds to the metal.
Importantly, the enforcement of the 3H4 conformation of the substrate at the TS by F659 not only provides a measure of TS stabilization by apposite CH-π interactions but also positions the C3-O3 and C4-O4 bonds of the ligand so as to further stabilize the partial positive charge at the TS through space electrostatically: in the language of preparative carbohydrate chemistry the substrate is super-armed. It has been broadly demonstrated that the greater chemical reactivity of galactopyranosides with respect to their gluco-isomers is mainly due to the through space stabilization of partial positive charge at the TS by the axial C4-O4 bond (Figure 5),18, 29, 61–65 as most apparent in Bols’ linear free energy relationships correlating rates of glycoside hydrolysis with pKa’s of analogously substituted polyhydroxypiperidines.29, 66, 67 This is directly analogous to the stabilization provided to the TS by side chains in the gg conformation (Figure 2). Extrapolating from this phenomenon, Bols and coworkers revealed dramatic rate increases in glycosidic bond hydrolysis when all C-O bonds are locked in pseudoaxial positions as illustrated by the ~450-fold rate increase seen with methyl 3,6-anhydro-β-D-glucopyranoside as compared to methyl α-D-glucopyransoide (Figure 5b).35 In preparative chemistry, Bols and others made use of this super-arming effect through the design and application of a series of conformationally distorted donors, with multiple pseudoaxial C-O bonds, that can be preferentially activated in the presence of related donors in the more usual 4C1 conformation (Figure 5c), albeit the situation is obscured in this case by the change in nature of the protecting groups.37 It appears that the GH47 mannosidases have evolved to distort their substrates away from the ground state conformation so as to position their C3-O3 and C4-O4 bonds in such a manner as to take advantage of super-arming and achieve higher levels of activity.
Figure 5.
(a) Impacts of C4 configuration on putative oxocarbenium stability (b) Relative rates of acidic hydrolysis of methyl glycosides with increasing axial character (c) Relative rates of activation of disarmed, armed, and super-armed glycosyl donors38
Enhancement of reactivity through conformational super-arming is not restricted to the GH47 α-mannosidases. In GH6 cellulases, a proximal tyrosine residue whose phenolic OH sterically prevents the substrate from taking up a 4C1 chair conformation forces the sugar into a 2E half chair or a 2SO skew boat with pseudoaxial substituents at C4 and C5 (Figure 6a), to which it also provides the more typical CH-π and/or hydrophobic stabilization.59 While the tenfold decrease in activity found by Larsson and coworkers with a Y73F mutant of T. fusca Cel6A can potentially by attributed to reduced stabilization of nascent positive charge at the anomeric center by the less electron rich arene, a Y73S mutant lacking the steric bulk of an aromatic ring results in a much larger 500-fold decrease in activity.68, 69 Crystal structures of the Y73S mutant bound to cellotetraose reveal that the pyranoside ring in the −1 site is held in the relaxed 4C1 conformation (Figure 6b), indicating that the tyrosine residue in the wild type enzyme increases reactivity by conformational arming of the substrate in the −1 site in addition to any stabilization it provides directly to the positive charge at the transition state. Interestingly, despite the significant destabilization afforded by placing the side chain above the pyranoside ring, GH6 endoglucanases hold the side chains of the ring undergoing hydrolysis in the gg conformation, thereby further maximizing reactivity.
Figure 6.
Partial crystal structures of (a) wild type T. fusca GH6 endoglucanase in complex with a thioglycoside (PDB ID 2BOD) and (b) Y73A T. fusca GH6 endoglucanase in complex with cellotetraose (PDB ID 2BOF) showing the pyranoside ring in the −1 site. Blue dashed lines designate hydrogen bonds.
In some cases, such as the GH81 and GH45 endoglucanases and the GH134 β-mannanases, enforcement of super-arming seems to be consistent throughout the family, with most or all available crystal structures binding the ligand in the more reactive conformation. Unlike the GH6 and 47 glycosidases, enforcement of the higher energy super-arming conformation by GHs 45, 81, and 134 is driven predominantly by H-bonding with the enzyme rather than by steric destabilization of the ground state conformation (Figure 7a–c).70–72 As with GH6, GHs 45 and 134 restrict their side chains to the most reactive gg conformation despite the destabilization afforded by placing the side chain directly above the ring. GH81 endoglucanases, on the other hand, avoid this steric penalty and hold their side chains in the gt conformation, constituting one of the exceptions discussed in our earlier analysis of enforced side chain conformations.32 In other families, only one of the crystal structures shows binding of the substrate in the super-armed conformation. For example, while most GH22 lysozymes bind their substrates in the more relaxed 4C1 conformation, one crystal structure of M. lusoria GH22 lysozyme reveals a tetrasaccharide-based unsaturated lactone-type inhibitor bound in a 5E conformation with pseudoaxial bonds at C4 and C5 distinct from the free solution E5 conformation73 of such lactones, indicating super-arming of the natural substrate (Figure 7d). Likewise, one crystal structure of a GH48 glucosidase holds a cellobio-derived isofagomine in the inverted 1C4 chair with the side chain in the gt conformation (Figure 7e).
Figure 7.
Partial crystal structures of (a) P. chryosporum GH45 endoglucanase bound to cellopentaose (PDB ID 3X2M), (b) B. halodurans GH81 glucosidase bound to laminarin (PDB ID 5T4G), (c) Streptomyces sp. GH134 β-mannanase bound to mannotriose (PDB ID 5JU9), (d) M. lusoria GH22 lysozyme bound to a tetrasaccharide-based unsaturated lactone (PDB ID 3AYQ), and (e) B. pumilus GH48 endoglucanase cellobiose-derived isofagomine (PDB ID 5VMA). Blue dashed lines designate hydrogen bonds.
Finally, it is appropriate to compare the rate enhancement provided by super-arming with that achieved by GHs over the uncatalyzed hydrolysis in water. Thus, super-arming provides an ~400-fold enhancement in the rate of hydrolysis of methyl glucosides (Figure 5) corresponding to a reduction in activation energy of ~1.9 kcal.mol−1, whereas GHs are known to accelerate hydrolysis by a factor of 1015–1021 over the uncatalyzed reaction in water (19–29 kcal.mol−1).74 Accordingly, we estimate that conformational super-arming can provide as much as 10% of the rate enhancement in hydrolysis achieved by the GHs that employ it.
Conclusion
We show that GH47 mannosidases and several other GH families impose a super-armed conformation on their substrates to enhance catalysis. Some families, such as GHs 47 and 6, enforce this conformation through steric interactions that favor substrate conformations with pseudoaxial C-O bonds at the 3- and 4-positions as well as a pseudoaxial side chain, whereas other families such as GHs 81, 134, and 45 employ H-bonding as the primary conformation driving factor. As with our previous analyses of side chain conformation preferences, this work illustrates that Nature is yet again one step ahead, having evolved to take advantage of a phenomenon that chemists have only recently discovered and began to exploit.
Acknowledgments:
We thank the NIH (GM62160) for support. Graphics were generated using UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.
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