An Epoxide Intermediate in Glycosidase Catalysis
Authors: Sha Zhu
Jesús Jiménez-Barbero
Andrew R Lewis
Dan Lu
Gaetano Speciale
Yongmin Zhang
Zalihe Hakki
Lluís Raich
Andrew J Thompson
Víctor Rojas-Cervellera
Ganeko Bernardo-Seisdedos
Carme Rovira
Oscar Millet
Andrew J Bennet
Saeideh Shamsi Kazem Abadi
Matthieu Sollogoub
Natalia Sannikova
Lukasz F Sobala
Gideon J Davies
Spencer J Williams
Journal: ACS Central Science (2020)
Discipline: CHEMISTRY
Keywords: glycoside hydrolases; stereochemical retention; neighboring-group participation; enzymatic mechanism; mannose 2-hydroxyl
Affiliations: York Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom, School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia, Sorbonne Université, CNRS, Institut Parisien de Chimie Moléculaire, UMR 8232, 4 place Jussieu, 75005 Paris, France, Departament de Quı́mica Inorgànica i Orgànica (Secció de Quı́mica Orgànica) & Institut de Quı́mica Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Martı́ i Franquès 1, 08028 Barcelona, Spain, Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada, Department of Biochemistry and Molecular Biology, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada, Molecular Recognition and Host−Pathogen Interactions, CIC bioGUNE, Basque Research Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain, Ikerbasque, Basque Foundation for Science, Marıá Dıáz de Haro 3, 48013 Bilbao, Spain, Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluı́s Companys 23, 08010 Barcelona, Spain

Abstract:  Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E 3) conformation. Kinetic isotope effects (k cat/K M) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism. 

Retracted: No

PubPeer comments:
 The paper  An Epoxide Intermediate in Glycosidase Catalysis by Sobala et al (ACS Cent. Sci. 2020, 6, 760-770) has many issues and is certainly not a shut-and-open case . Problem one. Page 762-left states: Tyr252 is approximately 4 Au030a away from C1 in BxGH99, and mutagenesis of the equivalent residue in human endo-u03b1-mannosidase (39% sequence identity to BxGH99) gave a mutant enzyme that maintained its ability to perform transglycosylation, excluding the possibility that this residue provides nucleophilic catalysis.22 On the contrary. Iwamoto et al (ChemBioChem DOI:10.1002 cbic.201700111) have mutated Tyr323 in human ER endo-alpha-1,2-mannosidase (equivalent to Tyr252 in BxGH99) but not to alanine, instead the mutation is to histidine. The Tyr323His mutant indeed shows residual transglycosylation, but that is exactly what is expected if Tyr323 is the nucleophile being replaced by histidine which is also a nucleophile and is near-isosteric. Problem two. Page763-right states: The free energy landscape reconstructed from the simulation (Figure 3a,b) shows that the Michaelis complex evolves to the product via a 1,2-anhydro sugar intermediate. An energy barrier of u0394Gu2021 u2248 15 kcal mol between the Michaelis complex (MC) and the transition state (TS) leads to the formation of a 1,2-anhydro sugar (EPO; local minimum on the right-hand- side). The computed free energy barrier is in good agreement with the available kinetic data;16 the reaction is therefore both feasible and favored. The start of the proposed reaction to the 1,2-anhydro sugar intermediate is an unstrained chair situation. The end-situation is however an epoxide, which has a ring strain of about 27 kcal mol, e.g. for oxirane: https: www.sciencedirect.com topics chemistry oxirane The end-situation is thus for at least about 27 kcal mol higher in energy than the start-situation. The TS must therefore strongly resemble the 1,2-anhydro sugar intermediate, thus practically all of the epoxide ring-strain build-up happens en-route to the TS. The mannoside ring also has a ring strain build-up when starting from the ground state chair, as proposed for the BxGH99 mechanism, toward the half chair in the 1,2-anhydro sugar intermediate, but that is only a few kcal mol since the six-membered ring of epoxycyclohexane should have a comparable ring strain as cyclohexene. The combined ring strain build-up en-route to the TS then amounts to about 30 kcal mol, to which the bond-breaking bond-making barrier at the TS must be added -- summating to a vastly higher potential barrier in comparison to that of classical reaction mechanisms that normal glycoside hydrolases are able to catalyze. It is reasonable to assume that the reaction starts not from a chair but from a twist-boat, since the latter is about 5 kcal mol higher in energy and thus for the same amount closer to the TS: https: chem.libretexts.org Bookshelves Organic_Chemistry Supplemental_Modules_(Organic_Chemistry) Alkanes Properties_of_Alkanes Cycloalkanes Ring_Strain_and_the_Structure_of_Cycloalkanes Even then, this gain is absolutely not enough to compensate for the combined ring strain build-up toward the TS. Nevertheless, kcat of GH99 members have been measured to be within the normal range of classical-acting glycoside hydrolases. Unavoidably this leads to only one of these two conclusions: - Either: The ring strain of oxirane is incorrect and cannot me more than just a few kcal mol; - Or: The proposed GH99 mechanism with formation of a 1,2-anhydro sugar intermediate must be abandoned. It should never have been postulated in the first place. And the QM MM metadynamics method computing u0394Gu2021 u2248 15 kcal mol is hopelessly inadequate (off by way more than 20 kcal mol!) for extracting nature-reflecting information from glycoside hydrolases. Problem three. The free energy landscape in figure 3a shows both Michaelis complexes (MC) as well as both 1,2-anhydro entities (EPO) having a free energy close to zero kcal mol (the dark blue wells). In light of the above, this is impossible: the free energy difference between MC and EPO definitely has to be about 30 kcal mol, even when complexed within the enzyme; crystal structures of GH99 enzymes nowhere show something extra peculiar that can super-stabilize a 1,2-anhydro sugar --a ground state-- for an extra 30 kcal mol. Anything resulting from the QM MM metadynamics study, thus any figures and all of its discussions throughout the article, are therefore of questionable scientific value. Problem four. Several retaining glycoside hydrolases have been trapped by sugar analogs bearing an epoxide; that something similar can occur on BxGH99 with the beta-1,2-epoxide 1 is not exceptional. The problem however is with the unreactive alpha-1,2-epoxide 2. Page 765-left states: The lack of reactivity of u03b1-1,2-epoxide 2, which stands in contrast to the reactivity observed for u03b2-1,2-epoxide 1, is consistent with stereochemical specificity of GH99 enzymes and provides evidence that there is not a nucleophilic enzymatic residue situated u201caboveu201d the sugar ring. This is radically not true. A nucleophilic attack on C1 of the beta-1,2-epoxide 1 will directly result in the ground state chair, whereas a nucleophilic attack on C1 of the alpha-1,2-epoxide 2 must lead to a twist-boat conformation. Only an attack on C2 of the alpha-1,2-epoxide 2 will directly result in a chair, but C2 will be unreachable for a nucleophile residue that is enzyme-positioned in order to attack C1. Therefore, the alpha-1,2-epoxide 2 is expected to remain intact even in the presence of a nucleophilic residue that is perfectly enzyme-positioned above C1 of the ring. See for a similar example here: https: www.chemtube3d.com epoxide_opening_chair . Quote of the last sentence therein, dealing with the same electronic situation as for a nucleophile attacking C1 of the alpha-1,2-epoxide 2: Nucleophilic attack at the other carbon would give a diaxial twist boat conformation, which could twist to give a fully equatorial chair product, but the kinetic barrier to forming the high energy, diaxial twist boat intermediate is too high for this to happen. That is a well-understood general phenomenon for reactions on sp2-sp2-like situations such as an epoxide, a double bond, or (!!) the electronically equivalent oxocarbenium ion, within six-membered rings: the substitution occurs by the route that directly gives the chair conformation, not by the route that gives a twist-boat. Which leads to problem five. It is then also expected that many classical mechanism-acting --passing through oxocarbenium ion-like transition states-- six-ring glycoside hydrolases, if not everyone of them, will have itineraries that directly give a chair at the end of the reaction, and will start with a higher-energy twist-boat at the beginning of the reaction. Likewise, twist-boat to twist-boat itineraries are unlikely, since also these pass through an oxocarbenium ion-like transition state after which this should collapse to a chair -- although such itineraries have already been attributed to many glycoside hydrolases, notably anointed with  evidence from the same QM MM metadynamics method. And back to problem one. A logical and sensible option is that Tyr252 is the actual nucleophile for BxGH99 --and for all GH99 members the equivalent tyrosine-- within a classical-type double displacement reaction itinerary. Re-stating from page 762-left: Tyr252 is approximately 4 Au030a away from C1 in BxGH99, ... which suggests a too far away distance, but is that always so? Is the tetrasaccharide permanently stuck in exactly the same position as soon as it gets in the enzyme s groove, just waiting there to react? The tetrasaccharide is observed as stuck in the BxGH99 crystal structure 6fwg allright, but that snapshot shows a chair in subsite -1, whereas one may expect a twist-boat for the start of the reaction. Within the time-frame of kcat a twist-boat in subsite -1 may occur many-many times at room temperature, but it may not appear often enough to be observed in the density of an X-ray experiment using a crystal in cryogenic state. When the twist-boat occurs in subsite -1, the tetrasaccharide in the enzyme s groove may shift somewhat to adapt to the difference in respective contacts, and the average distance of Tyr252 to C1 of the subsite -1 mannoside may very well become nucleophile-perfect. Plus, anyone who has seen a Molecular Dynamics video of a protein at 300K knows how frantically backbones and especially residues are moving, often over way more than one angstrom in the course of just a few nanoseconds. Imagine this during an immensely longer kcat time frame... then even the  unmoving Glu333 may sporadically position itself just right for nucleophilic attack at C1. A twist-boat at the start of the reaction within a GH47 ER alpha-1,2-mannosidase has been beautifully evidenced before, already 15 years ago, by Karaveg et al ( DOI: 10.1074 jbc.M500119200 ; https: www.jbc.org content 280 16 16197 ). And a tyrosine as a nucleophile within a classical reaction mechanism is not exceptional, as this is clearly seen in sialidases neuraminidases. Then there is Occam s razor. , 
 One more thought. Suppose it really is so --it is not-- that a GH99 enzyme can form the 1,2-anhydro sugar intermediate. Since this disaccharide species has only half of the ligand enzyme contacts than the minimal-natural tetrasasscharide substrate (actually less than half: the minus one subsite is preorganized to recognize the TS, not for recognizing a chair ground state), one expects this species to quickly dissipate from the enzyme. But the 1,2-anhydro sugar intermediate is not just an epoxide, it is a highly strained bicyclo [5,1,0] epoxy-acetal which must be very unstable in water. One can even have a rough estimate for how long it may survive, by comparing with thromboxane A2. Thromboxane A2 is a bicyclo [4,1,1] oxetane-acetal with a half-life in water of 32 seconds: https: pubs.acs.org doi 10.1021 acscentsci.0c00310 The ring strain of oxetane is about 25 kcal mol whereas that of oxirane is about 27 kcal mol: https: pubs.acs.org doi pdf 10.1021 acs.chemrev.6b00274 Therefore one may expect a bicyclo [5,1,0] epoxy-acetal to have a comparable, if not shorter, survival-time in water as thromboxane A2. The beta-1,2-epoxide 1 is a carba-analog and is a relatively stable molecule in water, for which a very slow transglycosylation by epoxide-ring opening can be catalyzed by a GH99 enzyme. But it should be clear from above that transglycosylation using an alpha-fluoro disaccharide (as e.g. in the Iwamoto et al 2017 paper, ChemBioChem DOI:10.1002 cbic.201700111 ) is unlikely with a GH99 epoxide mechanism, because the formed bicyclo [5,1,0] epoxy-acetal intermediate will instead dissipate from the enzyme and will quickly react with water. For efficient transglycosylation with GH99 using an alpha-fluoro disaccharide, this has to occur through formation of a real glycosyl-enzyme intermediate, where the intermediate is physically attached to the enzyme while the entity in the plus subsite(s) dissipates and gets replaced by the acceptor molecule. Therefore, GH99 has to have a catalytic nucleophile residue. And therefore the epoxide mechanism is a fable. The many authors of this paper should explain how all of them have read the Iwamoto et al 2017 paper (as all authors should read and at least try to understand all references) without noticing that Tyr323 was mutated to histidine. The alternative is worse, i.e. that none of the authors know that histidine can act as a nucleophile. Why did no-one see that the QM MM-calculated FEL obviously makes no sense, although they knew the ring strain penalty for the formation of an epoxide? How come no-one could realize that the alpha-1,2-epoxide is expected to be unreactive, although the substitution-selectivity at an epoxide fused to a six-membered ring is actually just a simple example of stereoelectronic control? , 
 The shut and open case GH99 epoxide mechanism has many inconsistencies that falsify it. Here is another experimental one. Naupactus Verecundus argues that the 1,2-anhydro sugar intermediate is not an isolated epoxide but a strained bicyclo-[4,1,0] epoxy acetal (not [5,1,0]) that will hydrolyze fast, by analogy with the similarly strained thromboxane-A2 which is a bicyclo-[3,1,1] oxetane acetal with only 32 seconds half-time in water. However, a bicyclo-[4,1,0] epoxy acetal and a bicyclo-[3,1,1] oxetane acetal are two different functionalities, for which one can only tentatively estimate the instability of the former by comparing to that of the latter. The bicyclo-[4,1,0] epoxy acetal intermediate is not covalently tethered to the enzyme, and, missing over half of the tetrasaccharide minimal substrate interactions, will indeed quickly depart from the active site. GH99 enzymes efficiently transglycosylate, and therefore the intermediate must be relatively stable in water, so that it can accumulate to a concentration that allows for sufficient active site re-occupancy, where the enzyme-catalyzed transglycosylation then occurs. It follows from the above that the bicyclo-[4,1,0] epoxy acetal intermediate will accumulate in solution and then slowly-gradually react with water or an alcohol reactant. This can easily be checked by following the GH99-catalyzed reaction with 1H-NMR: it will show the appearance and gradual disappearance of the intermediate, which can be identified unambiguously from its C1-hydrogen singlet resonance signal. This experiment has already been done, establishing the retaining mechanism of GH99 enzymes: https: www.pnas.org content pnas 109 3 781.full.pdf ; see the spectra in supplementary information Fig. 4: https: www.pnas.org content pnas suppl 2012 01 04 1111482109.DCSupplemental pnas.1111482109_SI.pdf?targetid=SF4 . The 1H-NMR experiment shows a clean hydrolysis in total absence of an intermediate. The bicyclo-[4,1,0] epoxy acetal should have been observed, but since it was not, then it must have a life-time in water that is way too short to allow transglycosylation. This contradicts the established fact that GH99 enzymes do transglycosylate. Vastly more logical is that the bicyclo-[4,1,0] epoxy acetal is simply not there at all, and that GH99 enzymes use a classical two-step mechanism passing a glycosyl-enzyme intermediate. ,