Carbohydrate Research xxx (2014) xxx–xxx
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (QM/MM) methods Igor Tvaroška ⇑ Department of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia Department of Chemistry, Faculty of Natural Sciences, Constantine the Philosopher University, SK-949 74 Nitra, Slovakia
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 12 June 2014 Accepted 16 June 2014 Available online xxxx Keywords: Glycosyltransferases Glycosyl transfer Catalytic mechanism Computational modeling QM/MM methods Nucleophilic substitution
a b s t r a c t Glycosyltransferases catalyze the formation of glycosidic bonds by assisting the transfer of a sugar residue from donors to specific acceptor molecules. Although structural and kinetic data have provided insight into mechanistic strategies employed by these enzymes, molecular modeling studies are essential for the understanding of glycosyltransferase catalyzed reactions at the atomistic level. For such modeling, combined quantum mechanics/molecular mechanics (QM/MM) methods have emerged as crucial. These methods allow the modeling of enzymatic reactions by using quantum mechanical methods for the calculation of the electronic structure of the active site models and treating the remaining enzyme environment by faster molecular mechanics methods. Herein, the application of QM/MM methods to glycosyltransferase catalyzed reactions is reviewed, and the insight from modeling of glycosyl transfer into the mechanisms and transition states structures of both inverting and retaining glycosyltransferases are discussed. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Glycan structures of N- and O-linked glycoproteins are at the center of diverse biological processes ranging from cell recognition, signaling, infection, and adhesion to cellular development. Glycosylation of proteins represents one of the most abundant posttranslational modifications and proceeds in a stepwise manner. A rich repertoire of biosynthesized glycans depends on the expression and specificity of carbohydrate acting enzymes, such as glycoside hydrolases and glycosyltransferases. Structural variations in these glycans are associated with many physiological and pathological cell processes, and it is assumed that an intervention of their biosynthesis may have therapeutic potential. The understanding of the catalytic mechanisms used by these enzymes is, therefore, of high interest. Complementary to experimental investigations, the QM/MM computational method is very useful tool for understanding of mechanistic strategy used by glycosyltransferases at the atomic level. Most importantly, these methods can directly determine the transition state (TS) structure, which is essential for the development of transition state analog inhibitors or drug design. ⇑ Tel.: +421 2 5941 0323; fax: +421 2 5941 0222. E-mail address: [email protected]
Glycosyltransferases (GTs, a general nomenclature for glycosyltransferases is EC 2.4.x.y) are carbohydrate acting enzymes that transfer glycosyl residues from activated donor molecules to acceptor molecules.1–4 For example, Leroir glycosyltransferases use sugar nucleotides such as, UDP-GlcNAc, UDP-Gal, and GDP-Man as a glycosyl donor. The acceptor substrates of glycosyltransferases(GTs) are carbohydrates, proteins, nucleic acids, and other molecules. In the catalytic reaction, the transfer of a monosaccharide residue can be considered as a nucleophilic displacement of the functional group (e.g., UDP) at the anomeric carbon C1 of the donor by the hydroxyl group of the acceptor. Functionally, glycosyltransferases are separated into ‘inverting’ or ‘retaining’ enzymes (Scheme 1). In inverting GTs, the result of the catalytic reaction is the inverted configuration at the anomeric carbon C1 (a ? b), whereas in retaining glycosyltransferases, the configuration at the C1 atom remains the same as in the donor (a ? a). Some glycosyltransferases that are metal ion-dependent, require a divalent cation for the catalytic reaction, others are metal ion-independent. Several reviews of mechanistic and structural studies of glycosyltransferases have been published.5–19 This mini-review focuses on recent advances in atomistic insight on the catalytic mechanism deciphered by QM/MM molecular modeling methods. Workers have used Density Functional Theory (DFT)
http://dx.doi.org/10.1016/j.carres.2014.06.017 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
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I. Tvaroška / Carbohydrate Research xxx (2014) xxx–xxx O
O
HO O
R2
Inverting GT (β linkage)
HO
+
O HO
P O R1 O-
O O
P O
R1
O-
(donor) (α linkage)
+
R2
OH
(acceptor)
O
Retaining GT (α linkage)
+
HO
O HO
P O
R1
O-
O
R2 Scheme 1. Reaction schemes depicting reaction mechanism of inverting and retaining Leroir glycosyltransferases. The R1 and R2 groups indicate the remaining part of nucleoside and the transferred acceptor, respectively.
for the QM part of these methods because of its speed relative to other post-Hartree Fock (HF) methods, and improved accuracy compared to HF methods themselves.
first structures of the transition state models for the catalytic reactions. These results, however, also revealed some limitations of this approach and it became apparent that relatively fine structural details of the model significantly affect the potential energy surface and may lead to different mechanisms. Therefore, it was clear that to understand all details of the catalytic mechanism of GTs it is necessary to take into account effects imposed by a whole protein environment, for example, by using QM/MM methods. A main complication for QM/MM studies of glycosyltransferases is the lack of structural data for the ternary complex between the particular glycosyltransferase and its natural substrates. Usually, only binary complexes of the GT are available with either the donor or acceptor.
2. Inverting glycosyltransferases The earliest molecular modeling studies of a catalytic mechanism of inverting glycosyltransferases used cluster models.20–23 In cluster approaches, the catalytic mechanism is investigated by high level quantum chemical methods,24 with models containing only a truncated part of the enzyme active site without a more complex enzymatic environment. The foremost model20 was based on the crystal structures of glycoside hydrolases, assuming an analogy between mechanisms of both enzyme groups. When the X-ray structures of glycosyltransferases became available, they were utilized for building truncated models.21 Though the cluster approach has clear limits, the results reproduced the available experimental data well. The calculations predicted, contrary to the glycoside hydrolases, that only one catalytic base is required for the catalytic reaction of glycosyltransferases. The results also predicted a direct displacement SN2-like reaction for inverting glycosyltransferases (Scheme 2) with the active site amino acid side chain functioning as a catalytic base. These findings are supported by all experimental data on glycosyltransferases so far. Furthermore, the calculations provided the
2.1. Metal-ion dependent glycosyltransferases The first QM(DFT)/MM studies on the catalytic mechanism of glycosyltransferases were carried out on the enzymes N-acetylglucosaminyltransferase I (GnT-I)25 and b-1,4-galactosyltransferase-1 (b4Gal-T1).26 These enzymes represent metal-ion dependent inverting GT-A fold glycosyltransferases and need a metal cofactor (usually Mn2+) for their activity. This divalent cation is coordinated by the so-called Asp-X-Asp (DXD) motif. GnT-I is involved in the formation of the hybrid and complex N-glycans by transferring the GlcNAc residue from UDP-GlcNAc to the C2-hydroxyl groups of the trimannosidic core of the
S N2-like mechanism GT
GT
:B
GT
:B
H
:BH
H O
O
O
R2
O HO O O
O
O
O-
O P O R1 O-
R2
+
HO
P O R1 O-
R2
HO
O -
O
P O
R1
O-
Transition state Scheme 2. A reaction scheme depicting reaction mechanism of inverting glycosyltransferases.
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Man5GlcNAc2 oligosaccharide (Scheme 3a). Since the reported crystal structures27 of the enzyme contained only the donor, the model was built by docking the trimannosyl Man-a1-3-(3, 6-OMe-Man-a1-6)-Manb oligosaccharide representing the acceptor into the X-ray structure of the binary complex of GnT-I with UDP-GlcNAc. In the QM/MM method,25 the entire ternary complex of GnT-I with donor, acceptor, and metal cofactor (Mn2+) consisted of 5721 atoms and was divided into QM and MM parts. The QM reaction site region (88 atoms) contained all residues relevant for the catalytic reaction (i) the UDP-GlcNAc portion of the sugardonor molecule, (ii) the Man-a1-3-mannose residue of the trisaccharide-acceptor, (iii) the catalytic base—aspartate D291, (iv) the divalent metal cofactor Mn2+ fully coordinated by three water molecules, and aspartate D213. The remaining part of the substrates and the enzyme, altogether 5633 atoms, were included in the MM region. The calculated reaction path supported a concerted SN2-type mechanism with the activation barrier of 19 kcal mol 1 and correlated well with available experimental data. The TS structure is characterized by asynchronous bond breaking of the C1–O1 (2.54 Å) bond and formation of the C1–O2 bond (1.91 Å) and is supported by experimental values of KIE’s.28,29 Simultaneously, the nucleophile O2 oxygen is deprotonated by the catalytic base D291 and a distorted ring shape with an almost planar arrangement around the anomeric carbon and a rotation of the diphosphate group. The overall effect of the protein environment on reaction energetics was found to be quite powerful, decreasing the overall reaction barrier by an estimated 9 kcal mol 1. This further supports the importance of including the protein environment in calculations of the enzymatic reactions. The inverting enzyme b4Gal-T1, in the absence of a-lactalbumin, catalyzes the transfer of the Gal residue from UDP-Gal to the O4 oxygen of GlcNAc in the presence of a Mn2+ metal ion (Scheme 4a). Biochemical analysis suggested Asp318 as the catalytic base and supported a direct displacement SN2-type mechanism.30–32
The structures of two binary complexes31,32 with UDP-Gal and GlcNAc were aligned to generate a ternary complex of the catalytic domain of Gal-T1 with its native substrates for an investigation using a QM(DFT)/MM method.26 The computational model of b4Gal-T1 contained 4529 atoms and was divided into QM and MM parts. The QM subsystem was quite large (253 atoms), consisting of the acceptor and donor substrates, the metal cofactor, and the side chains of 11 amino acids that influence the enzymatic reaction. The MM region was composed of the remaining 4274 enzyme atoms. The results support a concerted SN2-type mechanism for b4Gal-T1. The TS structure is similar to that of GnT-I, though the lengths of the formed and breaking bond are reversed. It is characterized by the 2.09 Å length for the breaking C1–O1 bond and the distance of 2.70 Å for the newly created C1–O4 bond. In TS, the transferred galactopyranose ring was distorted from the chair form with almost planar arrangement around the anomeric carbon and nearly perpendicular arrangement of both the leaving and attacking groups. Formation of the TS is accompanied by a rotation of the diphosphate group, which together with the movement of the C1 atom toward the nucleophilic oxygen of the acceptor smooth the progress of the enzymatic reaction. Since a similar movement of the diphosphate group was found for both GnT-I and b4Gal-T1, it was suggested that this is a distinguishing feature for the SN2 mechanism of inverting glycosyltransferases. The calculated barrier of 15 kcal mol 1 was consistent with the experimentally observed values. The QM(DFT)/MM calculations on both enzymes supported a concerted SN2-type mechanism for inverting metal-ion dependent GT-A fold glycosyltransferases. The structural analyses also revealed that GnT-I and b4Gal-T1 employed very similar strategies to assist the catalytic reaction. The low-barrier hydrogen bonds between the acceptor nucleophilic hydroxyl group and the carboxylate oxygen of the catalytic base that facilitates nucleophilic attack were observed in both glycosyltransferases. Moreover, it was found that in both enzymes the interaction between the HO OH OH O
N H
HO
OH O O
OMan 3 O
HO O
GlcNAc2 O
Asn-X
O
(a)
O O NH O P O P O O O- O-
O
OH
OGT, Ser/Thr
O
HO HO
NH HC CH2 C O
O
NH
(b)
O
G C2
OH OH
al ,G nT
UDP-GlcNAc (donor)
O
M
NH N
M an
HO HO
O O
G nT - I,
OH
5G lc n 2+ NA c
2-
As n
-X
HO HO
O
G
HO OH
X cNA al
OH O
N H
HO HO OH
(c)
O O
O HO
O
O
O Ser/Thr
NH OH O
Scheme 3. A schematic diagram of the enzymatic reaction catalyzed by (a) GnT-I, (b) OGT, and (c) C2GnT.
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OH
HO OH
O
O O HO OH
(a)
OH NAc
OH O O O P O P O OO-
O
O
HO OH
α3GalT, GalGlcNAc-X Mn
O OH
O
OH NAc
OH
OH O OH HO
O
O O HO OH
O
HO HO
(b)
OH
HO OH
HO
2+
X cNA lc G al 2+ ,G n M tC Lg
OH OH UDP-Gal (donor)
-T 1
NH N
G al
HO
M
O O
β4
HO OH
,G n 2+ lcN
A cX
HO
HO
(c)
O
O O HO OH
OH NAc
Scheme 4. A schematic diagram of the enzymatic reaction catalyzed by (a) b4Gal-T1, (b) a3GalT, and (c) LgtC.
acceptor’s hydroxyl group HO6 and b-phosphate oxygen stabilizes the building of a negative charge on the b-pyrophosphate. However, whereas in GnT-I this interaction is direct, in b4Gal-T1 this interaction is mediated by water molecules. The importance of the above interactions is supported by the experimental data on GnT-I that showed that removal of the C6 hydroxyl group decreases the enzyme activity and that an O6 methylated acceptor is tolerated as a substrate but has no catalytic activity.33 2.2. Metal-ion independent glycosyltransferases Two metal-ion independent enzymes, one from the GT-B group (OGT)34 and one from the GT-A group (Core2)35 were studied using QM/MM methods. These enzymes are fully active in the absence of divalent metal ions and do not contain a metal-binding DXD sequence. The essential mammalian enzyme, O-linked b-N-acetylaminyltransferase (OGT), catalyzes the addition of N-acetylglucosamine from UDP-GlcNAc to the hydroxyl group of serine or threonine residue of a vast number of various protein substrates (Scheme 3b). There is a considerable amount of evidence suggesting that O-GlcNAcylation regulates a wide range of cellular processes and is implicated in diseases including diabetes, cancer, and neurodegeneration.36,37 The ternary complex between the truncated human OGT, the acceptor 4.5 tetratricopeptide repeat units and the hydrolyzed donor UDP was reported and Histidine 498, was proposed as the catalytic base.38 Unfortunately, the GlcNAc part of the donor was missing from the crystal structure of this complex. Therefore, a docking procedure was used to obtain the entire OGT-UDPGlcNAc-CKII peptide ternary complex and this structure was used for investigation of the catalytic mechanism of OGT by QM(DFT)/MM calculations.34 The enzyme-substrate’s system, altogether 11,524 atoms, was divided into two regions: the QM and the MM regions. The QM subsystem, containing 198 atoms, was made of complete donor UDP-GlcNAc, three full residues of the acceptor CKII peptide, containing nucleophile Ser21 and the side chains of the catalytic base His498 and six other amino acids relevant to catalytic activity. Also, three water molecules in the vicinity of UDPGlcNAc were added into the QM region. The MM region (11,326 atoms) was composed of the remaining OGT and CK-II peptide atoms, and 20 water molecules were presented in the model complex. The catalytic reaction mechanism was investigated by generating three two-dimensional reaction energy surfaces (PES)
using three reaction coordinates. These corresponded to the formation of a new glycosidic bond, the cleavage of the donor glycosidic bond and a transfer of the proton from the acceptor hydroxyl to a catalytic base. The calculated PES showed the presence of one reaction pathway with a single transition barrier in the central area of the map and on the diagonal going from the Michaelis complex to the products. The presence of only one transition barrier is a clear indication of a concerted SN2-like mechanism. An inspection of geometrical changes showed that the nucleophilic attack by OSer, the proton transfer to the catalytic base and the dissociation of the leaving group all occur almost simultaneously. The energy barriers of 15.6, 19.6, and 15.5 kcal mol 1 calculated at the B3LYP, MPW1K, and M06-2X levels respectively were similar to the activation barrier of 21 kcal mol 1 estimated from the measured reaction rate constants for OGT. The calculations also revealed that the substrate acetamide stabilized the developing negative charge on the phosphate group by hydrogen bonding and thus facilitated the cleavage of the glycosidic linkage (Scheme 5). Such involvement of the acetamido group at the position adjacent to the anomeric bond in the catalytic mechanism was observed for the first time with glycosyltransferases, though OGA, a glycoside hydrolase involved in O-GlcNAcylation cycling uses an acetamido group as a nucleophile to cleave GlcNAc from serine/threonine.39 Though the results of these calculations were quite convincing, recently reported40,41 ternary complexes between OGT, a peptide
His Ser
NH N 1.68
O OH O
HO HO N O
H
1.92 O NH
3.11 H 1.79 O
N
O O P O P O OO-
O
O OH OH
Scheme 5. Schematic representation of the QM(DFT)/MM optimized TS structure for the reaction catalyzed by OGT. Numbers represent relevant distances in Å.31
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acceptor, and slowly transformed UDP-GlcNAc analog UDP-5-thio GlcNAc brought some doubt as to whether this is the correct mechanism. Interestingly, both these structural papers confirmed the role of the substrate acetamide in the transfer reaction, as predicted by QM/MM calculations.34 However, they proposed quite different mechanisms regardless of rather similar solved crystal structures (Scheme 6). In one of the proposed mechanisms,40 the a-phosphate is assumed to function as a base catalyst (Scheme 6a). On the other hand, in the second mechanism41 the a-phosphate was explicitly rejected as a candidate for the catalytic base. Instead, it was suggested that the proton be removed from the nucleophile via a chain of mediating water molecules in a Grotthuss mechanism possibly to D554 (Scheme 6b). However, the involvement of Asp554 is assumed unlikely by the authors of the first mechanism.40 An unanswered question is whether the replacement of the ring oxygen atom of donors by sulfur affects the mechanism of OGT. There are some experimental indications that modified 5-thio donors may influence the catalytic mechanism of inverting glycosyltransferases.42 Nevertheless, there are three different mechanisms proposed, and further experimental and theoretical studies are certainly needed to clarify this issue. The Golgi enzyme Core 2 b1,6-GlcNAc-transferase (C2GnT) is glycosyltransferase of the GT-A fold that catalyze the formation of the b1,6 linkage between GlcNAc and GalNAc of the core 1 structure, thus leading to the core 2 structure. Contrary to what was expected for glycosyltransferases with the GT-A fold, C2GnT is a metal ion-independent enzyme. Since the reported crystal structure43,44 of C2GnT is a binary complex with the acceptor Galb13-GalNAc, the ternary complex for QM(DFT)/MM calculations35 was constructed by docking the UDP donor into the active site of this binary complex. The structural model used for calculations contained 6120 atoms with 206 atoms in the QM subsystem. A detailed description of the reaction paths was obtained from two-dimensional potential energy maps considering three reaction coordinates that represented the formation of a new b1,6 glycosidic bond, the cleavage of the donor GlcNAc-phosphate glycosidic bond and transfer of a proton from the acceptor hydroxyl to a catalytic base. The results support the SN2-like mechanism and clearly show that the nucleophilic attack, dissociation of the C1–O1 glycosidic linkage and proton transfer from the nucleophile oxygen to the catalytic base all occur simultaneously. Similar to the situation for the inverting glycosyltransferase OGT,34 it was found that the hydrogen bond interaction between the HNAc proton of GlcNAc and the glycosidic oxygen O1 of the donor facilitate breaking the
glycosidic linkage and the withdrawal of the leaving group (UDP). The additional calculations led to a rough estimate of 11– 14 kcal mol 1 for the TS stabilization. Thus, despite the differences between their spatial folds, metal ion-independent inverting enzymes OGT and C2GnT used similar mechanistic strategies— the substrate assisted SN2-like mechanism. The structure of the transition state for the proposed reaction mechanism was similar to that calculated for OGT and was located at C1–O6 = 1.74 Å and C1–O1 = 2.86 Å. The calculated barrier was, depending on the DFT functional used, between 20 and 29 kcal mol 1, in agreement with barrier estimated from experimental measurements of rate constants (21 kcal mol 1). 3. Retaining glycosyltransferases Unlike the case with inverting GTs, an understanding of the catalytic mechanism of retaining GTs is not widely accepted with two mechanisms being debated.11,13 The first, based on analogy with glycoside hydrolases, is a double-displacement mechanism (Scheme 7) that requires two sequential SN2 steps and the formation of a covalently bound glycosyl-enzyme intermediate.45,46 For GTs lacking an appropriately positioned nucleophile in the active site an alternative SNi-like mechanism was proposed.47 3.1. Double-displacement mechanism The enzyme 3-a-D-galactosyltransferase (a3GalT) transfers galactose from UDP-Gal to the terminal N-acetyl lactosamine unit of glycans with retention of the anomeric configuration, producing the Gal-a1-3-Gal-b1-4-GlcNAc oligosaccharide structure present in most mammalian glycoproteins (Scheme 4b). The solved crystal structures of the catalytic domain of bovine a3GalT (substrate-free and substrate-bound complex) showed that a3GalT has a GT-A fold and the conserved residue E317 was proposed to function as the catalytic nucleophile involved in a double-displacement mechanism.48–50 Theoretical analyses23 of a truncated cluster model have shown that a3GalT proceeds via a double-displacement mechanism with the calculated reaction barrier of 14 kcal mol 1. This result was supported by a mutant rescue experiment45 that indicated the formation of a covalent intermediate with Glu317 functioning as the catalytic base. However, direct evidence for this intermediate has not been available. The role played by E317 in both a double-displacement and SNi mechanisms was investigated by static QM/MM calculations on
Asp C
O
O H
Ser
Ser OH O
O
HO HO
H
O NH
N O
O
H
OH
O
OO P O P O O O-
N O
H
O O
H O
O O P O P O OO-
N
O
O OH OH
OH OH
(a)
O NH
N
H
O
O
HO HO
(b)
Scheme 6. A schematic diagram of two proposed mechanisms for the reaction catalyzed by OGT. (a) The a-phosphate proposed as the catalytic base.38 (b) Asp 554 proposed as the catalytic base in Grotthus-like mechanism.37
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Double-displacement mechanism
GT
GT O
GT
:Nu
Nu
HO
:Nu
O
O
R2
O
HO
H
O O
P O
R1
HO
+
:B
O-
-
O
GT
O P O
R1
H
O
R2 B
O-
Scheme 7. A reaction scheme depicting a double replacement reaction mechanism of retaining glycosyltransferases.
multiple enzyme–substrate complexes extracted from MD simulations.51,52 The studied system of 12,694 atoms was divided into QM and MM regions with the QM region containing 84 atoms. For the double-displacement mechanism, the results are in qualitative agreement with previous calculations using the cluster model23 and support the role of UDP in deprotonating the nucleophile. For the SNi-like mechanism, the calculations supported SNi character for the transition state and UDP was found as the catalyst in the front-side attack. Interestingly, the authors, due to similar barriers, did not arrive at a conclusion as to which mechanism is the dominant one. However, their result suggested that interactions of the acceptor’s nucleophilic hydroxyl with phosphate oxygen would be relevant for the reaction. Another relevant conclusion is that E317 plays a crucial role in both mechanisms and, therefore, the interpretation of E317 mutation experiments is not simple. However, recent classical MD and QM(DFT)/MM metadynamic simulations53 clearly showed that a3GalT utilized a double displacement mechanism with E317 functioning as the catalytic base. The authors used crystal structures of two binary complexes48,50 of a3GalT to build ternary complex of a3GalT. This complex was relaxed using classical (MM based) MD simulation and then used for investigation of the catalytic mechanism by QM(DFT)/MM metadynamics. The transfer of the galactosyl residue was followed by means of two collective variables that describe the formation and dissociation of the relevant glycosidic linkages. The results revealed that the formation of the glycosyl-enzyme covalent intermediate is a dissociative process with an energy barrier of 23 kcal mol 1. The subsequent step (deglycosylation step), the breakup of the intermediate (breaking of the C1–OGlu linkage) and the formation of the product (formation of the C1–O3 linkage) required 13 kcal mol 1. This suggested that the formation of the glycosyl-enzyme covalent intermediate is the rate-limiting step. 3.2. SNi mechanism For retaining glycosyltransferases for which the catalytic base could not be identified in the catalytic site, a single nucleophilic displacement or an internal return mechanism SNi has been proposed (Scheme 8). This unique form of SN1 mechanism was first proposed for a lipopolysaccharide a-1,4-galactosyltransferase C (LgtC).47 LgtC is a retaining galactosyltransferase possessing a GT-A fold and is responsible for the transfer of a-galactose from UDP-Gal donor to a galactose residue of the terminal lactose moiety on the bacterial lipooligosaccharides (Scheme 4c). The X-ray structure of LgtC47 with Mn2+ and a nonreactive donor was solved in the absence and presence of the acceptor analog. Extensive theoretical calculations22,54,55 were used to gain some insights into characteristics of the enzymatic reaction catalyzed by LgtC. The DFT calculations of the truncated cluster model (136 atoms) predicted22 a one-step mechanism. In this mechanism, the nucleophile oxygen from the acceptor attacks the anomeric carbon of the donor UDP-Gal from
the side of the leaving group UDP, with simultaneous proton transfer to a phosphate oxygen. This suggested that the phosphate is functioning as a catalytic base to deprotonate the acceptor. This interaction fulfills two roles; it increases nucleophilicity of the acceptor hydroxyl oxygen and stabilizes the charge on the leaving phosphate group. These findings were recently confirmed by experimental data showing that retaining GTs of this kind use the leavinggroup phosphate oxygen as the catalytic base to deprotonate the acceptor nucleophilic oxygen.56,57 The calculations also showed that Gln189 was involved in the stabilization of the transition state by hydrogen bonding to the donor. The predicted structure of the transition state was unique and characteristic for the SNi mechanism. Recently, the catalytic mechanism of LgtC was also investigated by QM(DFT)/MM calculations.55 The results were consistent with the results on a truncated cluster model. In the calculations, the authors used the ternary complex of LgtC with UDP-Gal and lactose based on X-ray structures47 solvated with a 24 Å radius sphere of water molecules. The manganese ion was modeled by Mg2+, and the whole system consisted of 6728 atoms, with 101 atoms in the QM region. The calculations supported an SNi-like mechanism with highly dissociative character of the TS. The predicted QM(DFT)/MM barriers were in a reasonable agreement with the experimental value of 16 kcal mol 1.47,58 The free energy barrier was also determined with Umbrella Sampling Molecular Dynamics at a semiempirical level, a technique that forces the structure to visit high-energy conformation during the simulation instead of just the shapes that have high-probability at simulation temperatures. This method yielded a very high free energy barrier. Trehalose-6-phosphate synthase (OtsA) is a retaining glycosyltransferase of the GT-B fold. This enzyme catalyzes the transfer of a glucosyl residue from a donor substrate UDP-Glc to the 1-hydroxyl group of the acceptor substrate glucose-6-phosphate, yielding the product a,a-1,1-trehalose-6-phosphate (Scheme 9a). Experimental data on complexes between OtsA and pseudo-disaccharide inhibitors supported the SNi-like reaction in which the incoming nucleophile is deprotonated by departing phosphate functioning as a base.56,57 QM(DFT)/MM metadynamic simulations59 of the catalytic mechanism of OtsA are consistent with experimental data. In these calculations, the Michaelis complex and the products of this reaction were generated using the X-ray structure of the ternary complex of OtsA with UDP and validoxylamine-6-phosphate.56 QM/MM calculations used two collective variables to investigate glucose transfer by a metadynamic simulation. The first variable described the cleavage of the C1–O1 glycosidic linkage of the donor and the formation of the new glycoside linkage, while the second described the proton transfer from the acceptor to the phosphate group. The whole model contained 18,392 atoms with 72 atoms included in the QM region. The results supported the SNi-like reaction with an extremely short-lived intermediate. The calculations also showed an important role of
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S Ni-like mechanism GT
GT O
O
O O
HO
HO
R2
O
P O
H
O-
O
O
O
O
GT
R1
H
R2
HO
P O R1 -
+
O
O
R2 O
H
B
HO
P O
R1
O-
Transition state Scheme 8. A reaction scheme depicting a single step displacement SNi reaction mechanism of retaining glycosyltransferases.
OH HO HO
OH
O O
NH
OH O O O P O P O OO-
N
O
ΟtsA, Glc-6P
O
HO HO
OH OH HO O
2+
Mn
O
OH
(a)
O OP
OH OH UDP-Glc (donor) HO OH
O O
HO
OH NH
NH O O O P O P O O OO-
N
O
ppGalNAcT2, Ser/Thr 2+
Mn
HO HO
O
NH
O HC
CH2
NH
O
C O
(b)
O
OH OH UDP-GalNAc (donor)
HO HO
O
OH HO O
HO HO
HN H2N O O O P O P O OO
N
N N
OH HO O
Kre2p/Mnt1p, Man-X Mn2+
O HO
O
O
X
(c)
O OH OH
HO HO
GDP-Man (donor) Scheme 9. A schematic diagram of the enzymatic reaction catalyzed by (a) OtsA, (b) ppGalNAcT2, and (c) Kr2p/Mnt1.
the donor phosphate group. It serves as the catalytic base for the acceptor proton that assists in cleavage of the donor glycosidic bond. Two different groups recently studied the retaining enzyme polypeptide UDP-GalNAc transferase (ppGalNAcT2).60,61 This enzyme catalyzes the transfer of the GalNAc residue from the donor UDP-GalNAc to threonine/serine as a first step in mucin biosynthesis (Scheme 9b). The ppGalNAcT2 transferase is the metal dependent enzyme of the GT-A fold. In the first study, QM/MM calculations60 with the QM region containing 80 atoms and with the natural metal cofactor Mn2+ modeled by Mg2+. The calculated potential energy surface in the area corresponding to the energy maximum was very flat, and the transition state was not found. However, the calculations using QM(DFT)/MM method supported the SNi mechanism with the estimated reaction barrier at the M05-2X/TZVP//BP86/SVP level of 20 kcal mol 1. The authors also observed the hydrogen bond between the b-phosphate and the backbone amide group of the Thr acceptor that promoted the catalysis.
In the second investigation,61 the catalytic mechanism of this enzyme was studied by a combination of two different QM/MMbased approaches, namely a potential energy surface scan in two distance difference dimensions and minimum energy reaction path optimization using the Nudged Elastic Band method. In this study, the QM region contained 252 atoms. It was found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The calculated reaction barrier was 13.8 kcal mol 1, while the energy of the product complex was 6.7 kcal mol 1 lower compared to the reactant. The reaction energy profiles obtained using highlevel density functionals implied the presence of a very short-lived metastable oxocarbenium intermediate. The transition states for the proposed reaction mechanism were located at C1–O1 = 2.35 Å and C1–OThr = 2.97 Å for TS1 and C1–O1 = 3.60 Å and C1–OThr = 2.33 Å for TS2, respectively. It is noteworthy that the C1–OThr distance in TS2 is almost the same as the distance of the C1–O1 in the TS1. This observation supports the previously proposed concept18,57 of the two transition states involving each glycosidic bond being very similar, almost ‘mirror images’ of each other.
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The a-1,2-mannosyltransferase Kre2p/Mnt1p is a retaining glycosyltransferase of GT-A fold and is dependent on divalent metal Mn2+ for activity.62 Kre2p/Mnt1p transfers a-mannose from the donor GDP-Man to a terminal mannose moiety of the glycans (an acceptor substrate) by breaking the a-glycosidic bond in the donor substrate and forming a new a-1,2-glycosidic bond between the transferred mannose and the 2-OH group of the acceptor substrate (Scheme 9c). The catalytic mechanism of a-1,2-mannosyltransferase Kre2p/Mnt1p was modeled by QM(DFT)/MM methods. Kinetic and structural parameters of the transition states and intermediates, as well as kinetic isotope effects, were predicted.63 Since the presence of Mg2+, Ca2+ or Zn2+ significantly impaired the catalytic reaction,62,64 the effect of divalent cations Mn2+, Mg2+, Zn2+, and Ca2+ on the reaction’s chemical steps has been modeled. Contrary to the experimental data, the modeled reaction energy profiles of these divalent ions were similar. A similar conclusion was found in a previous study65 focused on modeling of dissociation of the C1–O1 glycosidic bond in galactosyl diphosphates in the presence of the Mn2+ and Mg2+ ions. The authors suggested that different catalytic properties of Mg2+, Zn2+ and Ca2+ ions compared to Mn2+ in Kre2p/Mnt1p could be due to thermodynamic rather than kinetic reasons. The catalysis in the presence of the metal ions is predicted as a step-wise SNi-like nucleophilic substitution reaction (DNint⁄ANàDhAxh) via oxocarbenium ion intermediates.63 A retaining a-(1,3)-galactosyltransferase (GTB) catalyzes the transfer of galactose from UDP-Gal to H antigen acceptor a-L-Fuc-(1,2)-b-D-Gal yielding the B antigen. GTB adopts a GT-A fold and its activity is dependent on the presence of manganese divalent ion Mn2+. The structure and behavior of GTB were comprehensively investigated.46 Hybrid QM(DFT)/MM calculations were used to elucidate the catalytic mechanism of this enzyme and its E303C mutant.66 Energetics of both a double-displacement and SNi-like mechanisms were compared. Calculations suggested that the SNi-like mechanism be preferred, though the double-displacement mechanism via a covalently bound glycosyl-enzyme intermediate is also possible. The energy barrier for the latter mechanism was calculated (18.3 kcal mol 1) to be higher compared to the SNi-like mechanism (10.1 kcal mol 1). Calculated asecondary kinetic isotope effects (a-KIE) of 1.28 and 1.29 for the wild enzyme and mutant, respectively predict a dissociative character of the transition state in agreement with experimentally measured a-KIE of other retaining glycosyltransferases.55,67 4. Transition state structure The structures of transition state models are an important outcome of the previously discussed QM and QM(DFT)/MM calculations of the catalytic mechanisms of glycosyltransferases. They may serve as a guide in structure-based drug design to developed transition state analog inhibitors for these enzymes. The calculations of the SN2 type mechanisms (inverting GTs and retaining GTs utilizing a double displacement mechanism) yielded to more than 20 different transition state models and provided information on possible structural variations of transition states (Scheme 10a). These structures had the following general characteristics: (a) the C1–O1 bond is elongated compared to the standard C–O bond length; (b) the lengths of a formed glycosidic linkage are also longer than the standard bond length; (c) the C1–O and C1–O1 bond lengths showed significant variations that can be as large as 1.0 and 1.7 Å, respectively; (d) both the forming and breaking bonds are oriented almost perpendicularly with respect to the plane defined by C2–C1–O5 atoms; (e) the anomeric carbon has a significant oxocarbenium character and has sp2 hybridization; (f) the transferred monosaccharide ring is flattened through C2–C1–O5–C5 and its conformation resembles a deformed chair/ envelope conformation. The calculated transition state structures
SNi-like mechanism
S N2-like mechanism Acceptor O
X
O
HO
1.6 - 2.7
2.6 - 3.4
1.6 - 3.2
O
O HO
1.6 - 3.2
O P
O
Donor
O-
P
R O
R
O-
O O
H
O-
X Acceptor
Donor
(a)
(b)
Scheme 10. A schematic representation of the transition state structures for glycosyltransferases utilizing (a) SN2 reaction mechanism and (b) SNi mechanism. Numbers represent relevant distances in Å.
also underline a significant contribution of the acceptor to transition state structure, and the development of stable transition state analogs as potent inhibitors of glycosyltransferases should, therefore, take acceptor features into account. The analysis of the transition state structures calculated for the SNi mechanism showed that the geometry of the transition state model is different (Scheme 10b). Though the TS structure differs from the transition state structures determined for glycosyltransferases using the SN2 mechanism, there are some similar features. The main difference compared to the SN2-type TS is that both a nucleophile oxygen and the phosphate leaving group oxygen are located on the a-face of the transferred monosaccharide. In contrast, values of the C1–O1 and C1–O distances are similar to those calculated for the SN2 mechanism, which indicate a weak bond order of the anomeric carbon of the donor to both the nucleophile and the leaving group. This supports an oxocarbenium character of TS. The ring conformation of the transferred monosaccharide is a distorted 4E envelope. Another interesting and unique feature of the SNi transition state is the location of the transferred proton. The nucleophile proton is sandwiched between the nucleophile oxygen and the phosphate oxygen. 5. Conclusion and outlook Hybrid QM(DFT)/MM methods are now regularly used to examine the catalytic mechanism of enzymes. The modeling studies have provided valuable information about the catalytic reaction of glycosyltransferases. The examples discussed in this minireview demonstrate that QM(DFT)/MM modeling is very useful in providing atomistic understanding of the reaction mechanism that is required for the rational design of specific and potent inhibitors/ drugs for this class of enzymes. In some cases, these calculations predicted features that were later confirmed by experiments and in many cases excellent agreement with experiments was observed. However, a permanent comparison of QM(DFT)/MM results to experimental data or to the models calculated by using high-level ab initio methods remains important. An investigation of the catalytic mechanism of glycosyltransferases using QM(DFT)/MM methods is not a trivial task. The QM(DFT)/ MM calculations require knowledge of the 3D structure, and one of the major issues is the structure of the Michaelis complex model. Unfortunately, the available structures of the ternary complexes between the glycosyltransferase, the donor, and the acceptor are limited. Usually, GT structures have only one active substrate (donor or acceptor) bound in the active site. Therefore, to build the atomic structure of Michaelis complex, docking methods or alignment of two different structures are used to accommodate a missing substrate into the active site. The structure of Michaelis complex
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model is the starting point for the QM(DFT)/MM calculations; therefore, it must be done very carefully. That is especially so, since even small deviations from the ‘accurate’ complex structure may fail to provide correct energies, and consequently, in some cases also the reaction mechanism. The next important issue is a separation of the whole system into the QM and MM regions, respectively. In particular, a proper definition of the size of the QM region and boundaries between QM and MM are essential and it has been suggested to have the QM region as large as possible.68–71 Additionally, based on a comparison of full QM and QM(DFT)/MM calculations, it was recommended to move the boundary between QM and MM regions at least one residue away from all active site residues.69 These requirements are not always possible to fulfill in the case of GTs, due to a large number of atoms in the active site (donor, acceptor, catalytic base, relevant amino acids, and metal ion). Nevertheless, it seems that the number of atoms in the QM region should be more than 100, and results with a smaller number of QM atoms should be considered with caution. The energy and structure of stationary points along reaction pathways are affected by the choice of functional and basis set used in the QM(DFT) method.72,73 Recently, it has been demonstrated that the calculated structure of TS depends on the simulation methods.74 Therefore, the method used should be suitable for a description of both carbohydrates and nucleophilic displacement reactions. Despite considerable progress in understanding of structure– function relationships of these enzymes, there are remaining challenges. Many glycosyltransferases exhibit conformational changes upon ligand binding, and the so-called ‘close’ and ‘open’ conformations were observed.30 The rate of these conformational changes often limits the overall rate of the enzymatic reactions. Recently, potent inhibitors of glycosyltransferases based on blocking the conformational change observed for these enzymes were developed.75,76 However, the character and role of these changes in the catalytic mechanism remains elusive and to get the complete picture of the reaction mechanism of GTs is one of the major challenges for the rational design of specific and potent inhibitors/ drugs for this class of enzymes. Without doubt, further developments in reliability and accuracy of QM(DFT)/MM methods will increase applications of these computational methods to study enzymatic reactions of carbohydrate processing enzymes. In silico experiments will more and more guide efforts in structure-based design of transition state analog inhibitors of glycoside hydrolases and glycosyltransferases. In general, the role of QM(DFT)/MM modeling methods in the area of glycoscience will unquestionably continue to grow. Acknowledgments This work was supported by Scientific Grant Agency of the Ministry of Education of Slovak Republic and Slovak Academy of Sciences (the project VEGA-02/0101/11), by the Slovak Academy of Sciences and National Science Council (Taiwan) bilateral project SAS-NSC JRP 2012/8, and the Research & Development Operational Programme funded by the ERDF (Centre of Excellence on Green Chemistry Methods and Processes, CEGreenI, Contract No. 26240120001, and Amplification of the Centre of Excellence on Green Chemistry Methods and Processes, CEGreenII, Contract No. 26240120025). References 1. Beyer, T. A.; Sadler, J. E.; Rearick, J. I.; Paulson, J. C.; Hill, R. L. Adv. Enzymol. 1981, 52, 23–175. 2. Schachter, H. Curr. Opin. Struct. Biol. 1991, 1, 755–765. 3. Kleene, R.; Berger, E. G. Biochim. Biophys. Acta 1993, 1154, 283–325. 4. New Comprehensive Biochemistry, Glycoproteins; Montreuil, J., Vliegenthart, J. F. G., Schachter, H., Eds.; Elsevier Science B.V.: Amsterdam, 1995; Vol. 29a.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Atomistic insight into the catalytic mechanism of glycosyltransferases by combined quantum mechanics/molecular mechanics (QM/MM) methods Igor Tvaroška ⇑ Department of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia Department of Chemistry, Faculty of Natural Sciences, Constantine the Philosopher University, SK-949 74 Nitra, Slovakia
a r t i c l e
i n f o
Article history: Received 23 May 2014 Received in revised form 12 June 2014 Accepted 16 June 2014 Available online xxxx Keywords: Glycosyltransferases Glycosyl transfer Catalytic mechanism Computational modeling QM/MM methods Nucleophilic substitution
a b s t r a c t Glycosyltransferases catalyze the formation of glycosidic bonds by assisting the transfer of a sugar residue from donors to specific acceptor molecules. Although structural and kinetic data have provided insight into mechanistic strategies employed by these enzymes, molecular modeling studies are essential for the understanding of glycosyltransferase catalyzed reactions at the atomistic level. For such modeling, combined quantum mechanics/molecular mechanics (QM/MM) methods have emerged as crucial. These methods allow the modeling of enzymatic reactions by using quantum mechanical methods for the calculation of the electronic structure of the active site models and treating the remaining enzyme environment by faster molecular mechanics methods. Herein, the application of QM/MM methods to glycosyltransferase catalyzed reactions is reviewed, and the insight from modeling of glycosyl transfer into the mechanisms and transition states structures of both inverting and retaining glycosyltransferases are discussed. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Glycan structures of N- and O-linked glycoproteins are at the center of diverse biological processes ranging from cell recognition, signaling, infection, and adhesion to cellular development. Glycosylation of proteins represents one of the most abundant posttranslational modifications and proceeds in a stepwise manner. A rich repertoire of biosynthesized glycans depends on the expression and specificity of carbohydrate acting enzymes, such as glycoside hydrolases and glycosyltransferases. Structural variations in these glycans are associated with many physiological and pathological cell processes, and it is assumed that an intervention of their biosynthesis may have therapeutic potential. The understanding of the catalytic mechanisms used by these enzymes is, therefore, of high interest. Complementary to experimental investigations, the QM/MM computational method is very useful tool for understanding of mechanistic strategy used by glycosyltransferases at the atomic level. Most importantly, these methods can directly determine the transition state (TS) structure, which is essential for the development of transition state analog inhibitors or drug design. ⇑ Tel.: +421 2 5941 0323; fax: +421 2 5941 0222. E-mail address: [email protected]
Glycosyltransferases (GTs, a general nomenclature for glycosyltransferases is EC 2.4.x.y) are carbohydrate acting enzymes that transfer glycosyl residues from activated donor molecules to acceptor molecules.1–4 For example, Leroir glycosyltransferases use sugar nucleotides such as, UDP-GlcNAc, UDP-Gal, and GDP-Man as a glycosyl donor. The acceptor substrates of glycosyltransferases(GTs) are carbohydrates, proteins, nucleic acids, and other molecules. In the catalytic reaction, the transfer of a monosaccharide residue can be considered as a nucleophilic displacement of the functional group (e.g., UDP) at the anomeric carbon C1 of the donor by the hydroxyl group of the acceptor. Functionally, glycosyltransferases are separated into ‘inverting’ or ‘retaining’ enzymes (Scheme 1). In inverting GTs, the result of the catalytic reaction is the inverted configuration at the anomeric carbon C1 (a ? b), whereas in retaining glycosyltransferases, the configuration at the C1 atom remains the same as in the donor (a ? a). Some glycosyltransferases that are metal ion-dependent, require a divalent cation for the catalytic reaction, others are metal ion-independent. Several reviews of mechanistic and structural studies of glycosyltransferases have been published.5–19 This mini-review focuses on recent advances in atomistic insight on the catalytic mechanism deciphered by QM/MM molecular modeling methods. Workers have used Density Functional Theory (DFT)
http://dx.doi.org/10.1016/j.carres.2014.06.017 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
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I. Tvaroška / Carbohydrate Research xxx (2014) xxx–xxx O
O
HO O
R2
Inverting GT (β linkage)
HO
+
O HO
P O R1 O-
O O
P O
R1
O-
(donor) (α linkage)
+
R2
OH
(acceptor)
O
Retaining GT (α linkage)
+
HO
O HO
P O
R1
O-
O
R2 Scheme 1. Reaction schemes depicting reaction mechanism of inverting and retaining Leroir glycosyltransferases. The R1 and R2 groups indicate the remaining part of nucleoside and the transferred acceptor, respectively.
for the QM part of these methods because of its speed relative to other post-Hartree Fock (HF) methods, and improved accuracy compared to HF methods themselves.
first structures of the transition state models for the catalytic reactions. These results, however, also revealed some limitations of this approach and it became apparent that relatively fine structural details of the model significantly affect the potential energy surface and may lead to different mechanisms. Therefore, it was clear that to understand all details of the catalytic mechanism of GTs it is necessary to take into account effects imposed by a whole protein environment, for example, by using QM/MM methods. A main complication for QM/MM studies of glycosyltransferases is the lack of structural data for the ternary complex between the particular glycosyltransferase and its natural substrates. Usually, only binary complexes of the GT are available with either the donor or acceptor.
2. Inverting glycosyltransferases The earliest molecular modeling studies of a catalytic mechanism of inverting glycosyltransferases used cluster models.20–23 In cluster approaches, the catalytic mechanism is investigated by high level quantum chemical methods,24 with models containing only a truncated part of the enzyme active site without a more complex enzymatic environment. The foremost model20 was based on the crystal structures of glycoside hydrolases, assuming an analogy between mechanisms of both enzyme groups. When the X-ray structures of glycosyltransferases became available, they were utilized for building truncated models.21 Though the cluster approach has clear limits, the results reproduced the available experimental data well. The calculations predicted, contrary to the glycoside hydrolases, that only one catalytic base is required for the catalytic reaction of glycosyltransferases. The results also predicted a direct displacement SN2-like reaction for inverting glycosyltransferases (Scheme 2) with the active site amino acid side chain functioning as a catalytic base. These findings are supported by all experimental data on glycosyltransferases so far. Furthermore, the calculations provided the
2.1. Metal-ion dependent glycosyltransferases The first QM(DFT)/MM studies on the catalytic mechanism of glycosyltransferases were carried out on the enzymes N-acetylglucosaminyltransferase I (GnT-I)25 and b-1,4-galactosyltransferase-1 (b4Gal-T1).26 These enzymes represent metal-ion dependent inverting GT-A fold glycosyltransferases and need a metal cofactor (usually Mn2+) for their activity. This divalent cation is coordinated by the so-called Asp-X-Asp (DXD) motif. GnT-I is involved in the formation of the hybrid and complex N-glycans by transferring the GlcNAc residue from UDP-GlcNAc to the C2-hydroxyl groups of the trimannosidic core of the
S N2-like mechanism GT
GT
:B
GT
:B
H
:BH
H O
O
O
R2
O HO O O
O
O
O-
O P O R1 O-
R2
+
HO
P O R1 O-
R2
HO
O -
O
P O
R1
O-
Transition state Scheme 2. A reaction scheme depicting reaction mechanism of inverting glycosyltransferases.
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Man5GlcNAc2 oligosaccharide (Scheme 3a). Since the reported crystal structures27 of the enzyme contained only the donor, the model was built by docking the trimannosyl Man-a1-3-(3, 6-OMe-Man-a1-6)-Manb oligosaccharide representing the acceptor into the X-ray structure of the binary complex of GnT-I with UDP-GlcNAc. In the QM/MM method,25 the entire ternary complex of GnT-I with donor, acceptor, and metal cofactor (Mn2+) consisted of 5721 atoms and was divided into QM and MM parts. The QM reaction site region (88 atoms) contained all residues relevant for the catalytic reaction (i) the UDP-GlcNAc portion of the sugardonor molecule, (ii) the Man-a1-3-mannose residue of the trisaccharide-acceptor, (iii) the catalytic base—aspartate D291, (iv) the divalent metal cofactor Mn2+ fully coordinated by three water molecules, and aspartate D213. The remaining part of the substrates and the enzyme, altogether 5633 atoms, were included in the MM region. The calculated reaction path supported a concerted SN2-type mechanism with the activation barrier of 19 kcal mol 1 and correlated well with available experimental data. The TS structure is characterized by asynchronous bond breaking of the C1–O1 (2.54 Å) bond and formation of the C1–O2 bond (1.91 Å) and is supported by experimental values of KIE’s.28,29 Simultaneously, the nucleophile O2 oxygen is deprotonated by the catalytic base D291 and a distorted ring shape with an almost planar arrangement around the anomeric carbon and a rotation of the diphosphate group. The overall effect of the protein environment on reaction energetics was found to be quite powerful, decreasing the overall reaction barrier by an estimated 9 kcal mol 1. This further supports the importance of including the protein environment in calculations of the enzymatic reactions. The inverting enzyme b4Gal-T1, in the absence of a-lactalbumin, catalyzes the transfer of the Gal residue from UDP-Gal to the O4 oxygen of GlcNAc in the presence of a Mn2+ metal ion (Scheme 4a). Biochemical analysis suggested Asp318 as the catalytic base and supported a direct displacement SN2-type mechanism.30–32
The structures of two binary complexes31,32 with UDP-Gal and GlcNAc were aligned to generate a ternary complex of the catalytic domain of Gal-T1 with its native substrates for an investigation using a QM(DFT)/MM method.26 The computational model of b4Gal-T1 contained 4529 atoms and was divided into QM and MM parts. The QM subsystem was quite large (253 atoms), consisting of the acceptor and donor substrates, the metal cofactor, and the side chains of 11 amino acids that influence the enzymatic reaction. The MM region was composed of the remaining 4274 enzyme atoms. The results support a concerted SN2-type mechanism for b4Gal-T1. The TS structure is similar to that of GnT-I, though the lengths of the formed and breaking bond are reversed. It is characterized by the 2.09 Å length for the breaking C1–O1 bond and the distance of 2.70 Å for the newly created C1–O4 bond. In TS, the transferred galactopyranose ring was distorted from the chair form with almost planar arrangement around the anomeric carbon and nearly perpendicular arrangement of both the leaving and attacking groups. Formation of the TS is accompanied by a rotation of the diphosphate group, which together with the movement of the C1 atom toward the nucleophilic oxygen of the acceptor smooth the progress of the enzymatic reaction. Since a similar movement of the diphosphate group was found for both GnT-I and b4Gal-T1, it was suggested that this is a distinguishing feature for the SN2 mechanism of inverting glycosyltransferases. The calculated barrier of 15 kcal mol 1 was consistent with the experimentally observed values. The QM(DFT)/MM calculations on both enzymes supported a concerted SN2-type mechanism for inverting metal-ion dependent GT-A fold glycosyltransferases. The structural analyses also revealed that GnT-I and b4Gal-T1 employed very similar strategies to assist the catalytic reaction. The low-barrier hydrogen bonds between the acceptor nucleophilic hydroxyl group and the carboxylate oxygen of the catalytic base that facilitates nucleophilic attack were observed in both glycosyltransferases. Moreover, it was found that in both enzymes the interaction between the HO OH OH O
N H
HO
OH O O
OMan 3 O
HO O
GlcNAc2 O
Asn-X
O
(a)
O O NH O P O P O O O- O-
O
OH
OGT, Ser/Thr
O
HO HO
NH HC CH2 C O
O
NH
(b)
O
G C2
OH OH
al ,G nT
UDP-GlcNAc (donor)
O
M
NH N
M an
HO HO
O O
G nT - I,
OH
5G lc n 2+ NA c
2-
As n
-X
HO HO
O
G
HO OH
X cNA al
OH O
N H
HO HO OH
(c)
O O
O HO
O
O
O Ser/Thr
NH OH O
Scheme 3. A schematic diagram of the enzymatic reaction catalyzed by (a) GnT-I, (b) OGT, and (c) C2GnT.
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OH
HO OH
O
O O HO OH
(a)
OH NAc
OH O O O P O P O OO-
O
O
HO OH
α3GalT, GalGlcNAc-X Mn
O OH
O
OH NAc
OH
OH O OH HO
O
O O HO OH
O
HO HO
(b)
OH
HO OH
HO
2+
X cNA lc G al 2+ ,G n M tC Lg
OH OH UDP-Gal (donor)
-T 1
NH N
G al
HO
M
O O
β4
HO OH
,G n 2+ lcN
A cX
HO
HO
(c)
O
O O HO OH
OH NAc
Scheme 4. A schematic diagram of the enzymatic reaction catalyzed by (a) b4Gal-T1, (b) a3GalT, and (c) LgtC.
acceptor’s hydroxyl group HO6 and b-phosphate oxygen stabilizes the building of a negative charge on the b-pyrophosphate. However, whereas in GnT-I this interaction is direct, in b4Gal-T1 this interaction is mediated by water molecules. The importance of the above interactions is supported by the experimental data on GnT-I that showed that removal of the C6 hydroxyl group decreases the enzyme activity and that an O6 methylated acceptor is tolerated as a substrate but has no catalytic activity.33 2.2. Metal-ion independent glycosyltransferases Two metal-ion independent enzymes, one from the GT-B group (OGT)34 and one from the GT-A group (Core2)35 were studied using QM/MM methods. These enzymes are fully active in the absence of divalent metal ions and do not contain a metal-binding DXD sequence. The essential mammalian enzyme, O-linked b-N-acetylaminyltransferase (OGT), catalyzes the addition of N-acetylglucosamine from UDP-GlcNAc to the hydroxyl group of serine or threonine residue of a vast number of various protein substrates (Scheme 3b). There is a considerable amount of evidence suggesting that O-GlcNAcylation regulates a wide range of cellular processes and is implicated in diseases including diabetes, cancer, and neurodegeneration.36,37 The ternary complex between the truncated human OGT, the acceptor 4.5 tetratricopeptide repeat units and the hydrolyzed donor UDP was reported and Histidine 498, was proposed as the catalytic base.38 Unfortunately, the GlcNAc part of the donor was missing from the crystal structure of this complex. Therefore, a docking procedure was used to obtain the entire OGT-UDPGlcNAc-CKII peptide ternary complex and this structure was used for investigation of the catalytic mechanism of OGT by QM(DFT)/MM calculations.34 The enzyme-substrate’s system, altogether 11,524 atoms, was divided into two regions: the QM and the MM regions. The QM subsystem, containing 198 atoms, was made of complete donor UDP-GlcNAc, three full residues of the acceptor CKII peptide, containing nucleophile Ser21 and the side chains of the catalytic base His498 and six other amino acids relevant to catalytic activity. Also, three water molecules in the vicinity of UDPGlcNAc were added into the QM region. The MM region (11,326 atoms) was composed of the remaining OGT and CK-II peptide atoms, and 20 water molecules were presented in the model complex. The catalytic reaction mechanism was investigated by generating three two-dimensional reaction energy surfaces (PES)
using three reaction coordinates. These corresponded to the formation of a new glycosidic bond, the cleavage of the donor glycosidic bond and a transfer of the proton from the acceptor hydroxyl to a catalytic base. The calculated PES showed the presence of one reaction pathway with a single transition barrier in the central area of the map and on the diagonal going from the Michaelis complex to the products. The presence of only one transition barrier is a clear indication of a concerted SN2-like mechanism. An inspection of geometrical changes showed that the nucleophilic attack by OSer, the proton transfer to the catalytic base and the dissociation of the leaving group all occur almost simultaneously. The energy barriers of 15.6, 19.6, and 15.5 kcal mol 1 calculated at the B3LYP, MPW1K, and M06-2X levels respectively were similar to the activation barrier of 21 kcal mol 1 estimated from the measured reaction rate constants for OGT. The calculations also revealed that the substrate acetamide stabilized the developing negative charge on the phosphate group by hydrogen bonding and thus facilitated the cleavage of the glycosidic linkage (Scheme 5). Such involvement of the acetamido group at the position adjacent to the anomeric bond in the catalytic mechanism was observed for the first time with glycosyltransferases, though OGA, a glycoside hydrolase involved in O-GlcNAcylation cycling uses an acetamido group as a nucleophile to cleave GlcNAc from serine/threonine.39 Though the results of these calculations were quite convincing, recently reported40,41 ternary complexes between OGT, a peptide
His Ser
NH N 1.68
O OH O
HO HO N O
H
1.92 O NH
3.11 H 1.79 O
N
O O P O P O OO-
O
O OH OH
Scheme 5. Schematic representation of the QM(DFT)/MM optimized TS structure for the reaction catalyzed by OGT. Numbers represent relevant distances in Å.31
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acceptor, and slowly transformed UDP-GlcNAc analog UDP-5-thio GlcNAc brought some doubt as to whether this is the correct mechanism. Interestingly, both these structural papers confirmed the role of the substrate acetamide in the transfer reaction, as predicted by QM/MM calculations.34 However, they proposed quite different mechanisms regardless of rather similar solved crystal structures (Scheme 6). In one of the proposed mechanisms,40 the a-phosphate is assumed to function as a base catalyst (Scheme 6a). On the other hand, in the second mechanism41 the a-phosphate was explicitly rejected as a candidate for the catalytic base. Instead, it was suggested that the proton be removed from the nucleophile via a chain of mediating water molecules in a Grotthuss mechanism possibly to D554 (Scheme 6b). However, the involvement of Asp554 is assumed unlikely by the authors of the first mechanism.40 An unanswered question is whether the replacement of the ring oxygen atom of donors by sulfur affects the mechanism of OGT. There are some experimental indications that modified 5-thio donors may influence the catalytic mechanism of inverting glycosyltransferases.42 Nevertheless, there are three different mechanisms proposed, and further experimental and theoretical studies are certainly needed to clarify this issue. The Golgi enzyme Core 2 b1,6-GlcNAc-transferase (C2GnT) is glycosyltransferase of the GT-A fold that catalyze the formation of the b1,6 linkage between GlcNAc and GalNAc of the core 1 structure, thus leading to the core 2 structure. Contrary to what was expected for glycosyltransferases with the GT-A fold, C2GnT is a metal ion-independent enzyme. Since the reported crystal structure43,44 of C2GnT is a binary complex with the acceptor Galb13-GalNAc, the ternary complex for QM(DFT)/MM calculations35 was constructed by docking the UDP donor into the active site of this binary complex. The structural model used for calculations contained 6120 atoms with 206 atoms in the QM subsystem. A detailed description of the reaction paths was obtained from two-dimensional potential energy maps considering three reaction coordinates that represented the formation of a new b1,6 glycosidic bond, the cleavage of the donor GlcNAc-phosphate glycosidic bond and transfer of a proton from the acceptor hydroxyl to a catalytic base. The results support the SN2-like mechanism and clearly show that the nucleophilic attack, dissociation of the C1–O1 glycosidic linkage and proton transfer from the nucleophile oxygen to the catalytic base all occur simultaneously. Similar to the situation for the inverting glycosyltransferase OGT,34 it was found that the hydrogen bond interaction between the HNAc proton of GlcNAc and the glycosidic oxygen O1 of the donor facilitate breaking the
glycosidic linkage and the withdrawal of the leaving group (UDP). The additional calculations led to a rough estimate of 11– 14 kcal mol 1 for the TS stabilization. Thus, despite the differences between their spatial folds, metal ion-independent inverting enzymes OGT and C2GnT used similar mechanistic strategies— the substrate assisted SN2-like mechanism. The structure of the transition state for the proposed reaction mechanism was similar to that calculated for OGT and was located at C1–O6 = 1.74 Å and C1–O1 = 2.86 Å. The calculated barrier was, depending on the DFT functional used, between 20 and 29 kcal mol 1, in agreement with barrier estimated from experimental measurements of rate constants (21 kcal mol 1). 3. Retaining glycosyltransferases Unlike the case with inverting GTs, an understanding of the catalytic mechanism of retaining GTs is not widely accepted with two mechanisms being debated.11,13 The first, based on analogy with glycoside hydrolases, is a double-displacement mechanism (Scheme 7) that requires two sequential SN2 steps and the formation of a covalently bound glycosyl-enzyme intermediate.45,46 For GTs lacking an appropriately positioned nucleophile in the active site an alternative SNi-like mechanism was proposed.47 3.1. Double-displacement mechanism The enzyme 3-a-D-galactosyltransferase (a3GalT) transfers galactose from UDP-Gal to the terminal N-acetyl lactosamine unit of glycans with retention of the anomeric configuration, producing the Gal-a1-3-Gal-b1-4-GlcNAc oligosaccharide structure present in most mammalian glycoproteins (Scheme 4b). The solved crystal structures of the catalytic domain of bovine a3GalT (substrate-free and substrate-bound complex) showed that a3GalT has a GT-A fold and the conserved residue E317 was proposed to function as the catalytic nucleophile involved in a double-displacement mechanism.48–50 Theoretical analyses23 of a truncated cluster model have shown that a3GalT proceeds via a double-displacement mechanism with the calculated reaction barrier of 14 kcal mol 1. This result was supported by a mutant rescue experiment45 that indicated the formation of a covalent intermediate with Glu317 functioning as the catalytic base. However, direct evidence for this intermediate has not been available. The role played by E317 in both a double-displacement and SNi mechanisms was investigated by static QM/MM calculations on
Asp C
O
O H
Ser
Ser OH O
O
HO HO
H
O NH
N O
O
H
OH
O
OO P O P O O O-
N O
H
O O
H O
O O P O P O OO-
N
O
O OH OH
OH OH
(a)
O NH
N
H
O
O
HO HO
(b)
Scheme 6. A schematic diagram of two proposed mechanisms for the reaction catalyzed by OGT. (a) The a-phosphate proposed as the catalytic base.38 (b) Asp 554 proposed as the catalytic base in Grotthus-like mechanism.37
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Double-displacement mechanism
GT
GT O
GT
:Nu
Nu
HO
:Nu
O
O
R2
O
HO
H
O O
P O
R1
HO
+
:B
O-
-
O
GT
O P O
R1
H
O
R2 B
O-
Scheme 7. A reaction scheme depicting a double replacement reaction mechanism of retaining glycosyltransferases.
multiple enzyme–substrate complexes extracted from MD simulations.51,52 The studied system of 12,694 atoms was divided into QM and MM regions with the QM region containing 84 atoms. For the double-displacement mechanism, the results are in qualitative agreement with previous calculations using the cluster model23 and support the role of UDP in deprotonating the nucleophile. For the SNi-like mechanism, the calculations supported SNi character for the transition state and UDP was found as the catalyst in the front-side attack. Interestingly, the authors, due to similar barriers, did not arrive at a conclusion as to which mechanism is the dominant one. However, their result suggested that interactions of the acceptor’s nucleophilic hydroxyl with phosphate oxygen would be relevant for the reaction. Another relevant conclusion is that E317 plays a crucial role in both mechanisms and, therefore, the interpretation of E317 mutation experiments is not simple. However, recent classical MD and QM(DFT)/MM metadynamic simulations53 clearly showed that a3GalT utilized a double displacement mechanism with E317 functioning as the catalytic base. The authors used crystal structures of two binary complexes48,50 of a3GalT to build ternary complex of a3GalT. This complex was relaxed using classical (MM based) MD simulation and then used for investigation of the catalytic mechanism by QM(DFT)/MM metadynamics. The transfer of the galactosyl residue was followed by means of two collective variables that describe the formation and dissociation of the relevant glycosidic linkages. The results revealed that the formation of the glycosyl-enzyme covalent intermediate is a dissociative process with an energy barrier of 23 kcal mol 1. The subsequent step (deglycosylation step), the breakup of the intermediate (breaking of the C1–OGlu linkage) and the formation of the product (formation of the C1–O3 linkage) required 13 kcal mol 1. This suggested that the formation of the glycosyl-enzyme covalent intermediate is the rate-limiting step. 3.2. SNi mechanism For retaining glycosyltransferases for which the catalytic base could not be identified in the catalytic site, a single nucleophilic displacement or an internal return mechanism SNi has been proposed (Scheme 8). This unique form of SN1 mechanism was first proposed for a lipopolysaccharide a-1,4-galactosyltransferase C (LgtC).47 LgtC is a retaining galactosyltransferase possessing a GT-A fold and is responsible for the transfer of a-galactose from UDP-Gal donor to a galactose residue of the terminal lactose moiety on the bacterial lipooligosaccharides (Scheme 4c). The X-ray structure of LgtC47 with Mn2+ and a nonreactive donor was solved in the absence and presence of the acceptor analog. Extensive theoretical calculations22,54,55 were used to gain some insights into characteristics of the enzymatic reaction catalyzed by LgtC. The DFT calculations of the truncated cluster model (136 atoms) predicted22 a one-step mechanism. In this mechanism, the nucleophile oxygen from the acceptor attacks the anomeric carbon of the donor UDP-Gal from
the side of the leaving group UDP, with simultaneous proton transfer to a phosphate oxygen. This suggested that the phosphate is functioning as a catalytic base to deprotonate the acceptor. This interaction fulfills two roles; it increases nucleophilicity of the acceptor hydroxyl oxygen and stabilizes the charge on the leaving phosphate group. These findings were recently confirmed by experimental data showing that retaining GTs of this kind use the leavinggroup phosphate oxygen as the catalytic base to deprotonate the acceptor nucleophilic oxygen.56,57 The calculations also showed that Gln189 was involved in the stabilization of the transition state by hydrogen bonding to the donor. The predicted structure of the transition state was unique and characteristic for the SNi mechanism. Recently, the catalytic mechanism of LgtC was also investigated by QM(DFT)/MM calculations.55 The results were consistent with the results on a truncated cluster model. In the calculations, the authors used the ternary complex of LgtC with UDP-Gal and lactose based on X-ray structures47 solvated with a 24 Å radius sphere of water molecules. The manganese ion was modeled by Mg2+, and the whole system consisted of 6728 atoms, with 101 atoms in the QM region. The calculations supported an SNi-like mechanism with highly dissociative character of the TS. The predicted QM(DFT)/MM barriers were in a reasonable agreement with the experimental value of 16 kcal mol 1.47,58 The free energy barrier was also determined with Umbrella Sampling Molecular Dynamics at a semiempirical level, a technique that forces the structure to visit high-energy conformation during the simulation instead of just the shapes that have high-probability at simulation temperatures. This method yielded a very high free energy barrier. Trehalose-6-phosphate synthase (OtsA) is a retaining glycosyltransferase of the GT-B fold. This enzyme catalyzes the transfer of a glucosyl residue from a donor substrate UDP-Glc to the 1-hydroxyl group of the acceptor substrate glucose-6-phosphate, yielding the product a,a-1,1-trehalose-6-phosphate (Scheme 9a). Experimental data on complexes between OtsA and pseudo-disaccharide inhibitors supported the SNi-like reaction in which the incoming nucleophile is deprotonated by departing phosphate functioning as a base.56,57 QM(DFT)/MM metadynamic simulations59 of the catalytic mechanism of OtsA are consistent with experimental data. In these calculations, the Michaelis complex and the products of this reaction were generated using the X-ray structure of the ternary complex of OtsA with UDP and validoxylamine-6-phosphate.56 QM/MM calculations used two collective variables to investigate glucose transfer by a metadynamic simulation. The first variable described the cleavage of the C1–O1 glycosidic linkage of the donor and the formation of the new glycoside linkage, while the second described the proton transfer from the acceptor to the phosphate group. The whole model contained 18,392 atoms with 72 atoms included in the QM region. The results supported the SNi-like reaction with an extremely short-lived intermediate. The calculations also showed an important role of
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S Ni-like mechanism GT
GT O
O
O O
HO
HO
R2
O
P O
H
O-
O
O
O
O
GT
R1
H
R2
HO
P O R1 -
+
O
O
R2 O
H
B
HO
P O
R1
O-
Transition state Scheme 8. A reaction scheme depicting a single step displacement SNi reaction mechanism of retaining glycosyltransferases.
OH HO HO
OH
O O
NH
OH O O O P O P O OO-
N
O
ΟtsA, Glc-6P
O
HO HO
OH OH HO O
2+
Mn
O
OH
(a)
O OP
OH OH UDP-Glc (donor) HO OH
O O
HO
OH NH
NH O O O P O P O O OO-
N
O
ppGalNAcT2, Ser/Thr 2+
Mn
HO HO
O
NH
O HC
CH2
NH
O
C O
(b)
O
OH OH UDP-GalNAc (donor)
HO HO
O
OH HO O
HO HO
HN H2N O O O P O P O OO
N
N N
OH HO O
Kre2p/Mnt1p, Man-X Mn2+
O HO
O
O
X
(c)
O OH OH
HO HO
GDP-Man (donor) Scheme 9. A schematic diagram of the enzymatic reaction catalyzed by (a) OtsA, (b) ppGalNAcT2, and (c) Kr2p/Mnt1.
the donor phosphate group. It serves as the catalytic base for the acceptor proton that assists in cleavage of the donor glycosidic bond. Two different groups recently studied the retaining enzyme polypeptide UDP-GalNAc transferase (ppGalNAcT2).60,61 This enzyme catalyzes the transfer of the GalNAc residue from the donor UDP-GalNAc to threonine/serine as a first step in mucin biosynthesis (Scheme 9b). The ppGalNAcT2 transferase is the metal dependent enzyme of the GT-A fold. In the first study, QM/MM calculations60 with the QM region containing 80 atoms and with the natural metal cofactor Mn2+ modeled by Mg2+. The calculated potential energy surface in the area corresponding to the energy maximum was very flat, and the transition state was not found. However, the calculations using QM(DFT)/MM method supported the SNi mechanism with the estimated reaction barrier at the M05-2X/TZVP//BP86/SVP level of 20 kcal mol 1. The authors also observed the hydrogen bond between the b-phosphate and the backbone amide group of the Thr acceptor that promoted the catalysis.
In the second investigation,61 the catalytic mechanism of this enzyme was studied by a combination of two different QM/MMbased approaches, namely a potential energy surface scan in two distance difference dimensions and minimum energy reaction path optimization using the Nudged Elastic Band method. In this study, the QM region contained 252 atoms. It was found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The calculated reaction barrier was 13.8 kcal mol 1, while the energy of the product complex was 6.7 kcal mol 1 lower compared to the reactant. The reaction energy profiles obtained using highlevel density functionals implied the presence of a very short-lived metastable oxocarbenium intermediate. The transition states for the proposed reaction mechanism were located at C1–O1 = 2.35 Å and C1–OThr = 2.97 Å for TS1 and C1–O1 = 3.60 Å and C1–OThr = 2.33 Å for TS2, respectively. It is noteworthy that the C1–OThr distance in TS2 is almost the same as the distance of the C1–O1 in the TS1. This observation supports the previously proposed concept18,57 of the two transition states involving each glycosidic bond being very similar, almost ‘mirror images’ of each other.
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The a-1,2-mannosyltransferase Kre2p/Mnt1p is a retaining glycosyltransferase of GT-A fold and is dependent on divalent metal Mn2+ for activity.62 Kre2p/Mnt1p transfers a-mannose from the donor GDP-Man to a terminal mannose moiety of the glycans (an acceptor substrate) by breaking the a-glycosidic bond in the donor substrate and forming a new a-1,2-glycosidic bond between the transferred mannose and the 2-OH group of the acceptor substrate (Scheme 9c). The catalytic mechanism of a-1,2-mannosyltransferase Kre2p/Mnt1p was modeled by QM(DFT)/MM methods. Kinetic and structural parameters of the transition states and intermediates, as well as kinetic isotope effects, were predicted.63 Since the presence of Mg2+, Ca2+ or Zn2+ significantly impaired the catalytic reaction,62,64 the effect of divalent cations Mn2+, Mg2+, Zn2+, and Ca2+ on the reaction’s chemical steps has been modeled. Contrary to the experimental data, the modeled reaction energy profiles of these divalent ions were similar. A similar conclusion was found in a previous study65 focused on modeling of dissociation of the C1–O1 glycosidic bond in galactosyl diphosphates in the presence of the Mn2+ and Mg2+ ions. The authors suggested that different catalytic properties of Mg2+, Zn2+ and Ca2+ ions compared to Mn2+ in Kre2p/Mnt1p could be due to thermodynamic rather than kinetic reasons. The catalysis in the presence of the metal ions is predicted as a step-wise SNi-like nucleophilic substitution reaction (DNint⁄ANàDhAxh) via oxocarbenium ion intermediates.63 A retaining a-(1,3)-galactosyltransferase (GTB) catalyzes the transfer of galactose from UDP-Gal to H antigen acceptor a-L-Fuc-(1,2)-b-D-Gal yielding the B antigen. GTB adopts a GT-A fold and its activity is dependent on the presence of manganese divalent ion Mn2+. The structure and behavior of GTB were comprehensively investigated.46 Hybrid QM(DFT)/MM calculations were used to elucidate the catalytic mechanism of this enzyme and its E303C mutant.66 Energetics of both a double-displacement and SNi-like mechanisms were compared. Calculations suggested that the SNi-like mechanism be preferred, though the double-displacement mechanism via a covalently bound glycosyl-enzyme intermediate is also possible. The energy barrier for the latter mechanism was calculated (18.3 kcal mol 1) to be higher compared to the SNi-like mechanism (10.1 kcal mol 1). Calculated asecondary kinetic isotope effects (a-KIE) of 1.28 and 1.29 for the wild enzyme and mutant, respectively predict a dissociative character of the transition state in agreement with experimentally measured a-KIE of other retaining glycosyltransferases.55,67 4. Transition state structure The structures of transition state models are an important outcome of the previously discussed QM and QM(DFT)/MM calculations of the catalytic mechanisms of glycosyltransferases. They may serve as a guide in structure-based drug design to developed transition state analog inhibitors for these enzymes. The calculations of the SN2 type mechanisms (inverting GTs and retaining GTs utilizing a double displacement mechanism) yielded to more than 20 different transition state models and provided information on possible structural variations of transition states (Scheme 10a). These structures had the following general characteristics: (a) the C1–O1 bond is elongated compared to the standard C–O bond length; (b) the lengths of a formed glycosidic linkage are also longer than the standard bond length; (c) the C1–O and C1–O1 bond lengths showed significant variations that can be as large as 1.0 and 1.7 Å, respectively; (d) both the forming and breaking bonds are oriented almost perpendicularly with respect to the plane defined by C2–C1–O5 atoms; (e) the anomeric carbon has a significant oxocarbenium character and has sp2 hybridization; (f) the transferred monosaccharide ring is flattened through C2–C1–O5–C5 and its conformation resembles a deformed chair/ envelope conformation. The calculated transition state structures
SNi-like mechanism
S N2-like mechanism Acceptor O
X
O
HO
1.6 - 2.7
2.6 - 3.4
1.6 - 3.2
O
O HO
1.6 - 3.2
O P
O
Donor
O-
P
R O
R
O-
O O
H
O-
X Acceptor
Donor
(a)
(b)
Scheme 10. A schematic representation of the transition state structures for glycosyltransferases utilizing (a) SN2 reaction mechanism and (b) SNi mechanism. Numbers represent relevant distances in Å.
also underline a significant contribution of the acceptor to transition state structure, and the development of stable transition state analogs as potent inhibitors of glycosyltransferases should, therefore, take acceptor features into account. The analysis of the transition state structures calculated for the SNi mechanism showed that the geometry of the transition state model is different (Scheme 10b). Though the TS structure differs from the transition state structures determined for glycosyltransferases using the SN2 mechanism, there are some similar features. The main difference compared to the SN2-type TS is that both a nucleophile oxygen and the phosphate leaving group oxygen are located on the a-face of the transferred monosaccharide. In contrast, values of the C1–O1 and C1–O distances are similar to those calculated for the SN2 mechanism, which indicate a weak bond order of the anomeric carbon of the donor to both the nucleophile and the leaving group. This supports an oxocarbenium character of TS. The ring conformation of the transferred monosaccharide is a distorted 4E envelope. Another interesting and unique feature of the SNi transition state is the location of the transferred proton. The nucleophile proton is sandwiched between the nucleophile oxygen and the phosphate oxygen. 5. Conclusion and outlook Hybrid QM(DFT)/MM methods are now regularly used to examine the catalytic mechanism of enzymes. The modeling studies have provided valuable information about the catalytic reaction of glycosyltransferases. The examples discussed in this minireview demonstrate that QM(DFT)/MM modeling is very useful in providing atomistic understanding of the reaction mechanism that is required for the rational design of specific and potent inhibitors/ drugs for this class of enzymes. In some cases, these calculations predicted features that were later confirmed by experiments and in many cases excellent agreement with experiments was observed. However, a permanent comparison of QM(DFT)/MM results to experimental data or to the models calculated by using high-level ab initio methods remains important. An investigation of the catalytic mechanism of glycosyltransferases using QM(DFT)/MM methods is not a trivial task. The QM(DFT)/ MM calculations require knowledge of the 3D structure, and one of the major issues is the structure of the Michaelis complex model. Unfortunately, the available structures of the ternary complexes between the glycosyltransferase, the donor, and the acceptor are limited. Usually, GT structures have only one active substrate (donor or acceptor) bound in the active site. Therefore, to build the atomic structure of Michaelis complex, docking methods or alignment of two different structures are used to accommodate a missing substrate into the active site. The structure of Michaelis complex
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model is the starting point for the QM(DFT)/MM calculations; therefore, it must be done very carefully. That is especially so, since even small deviations from the ‘accurate’ complex structure may fail to provide correct energies, and consequently, in some cases also the reaction mechanism. The next important issue is a separation of the whole system into the QM and MM regions, respectively. In particular, a proper definition of the size of the QM region and boundaries between QM and MM are essential and it has been suggested to have the QM region as large as possible.68–71 Additionally, based on a comparison of full QM and QM(DFT)/MM calculations, it was recommended to move the boundary between QM and MM regions at least one residue away from all active site residues.69 These requirements are not always possible to fulfill in the case of GTs, due to a large number of atoms in the active site (donor, acceptor, catalytic base, relevant amino acids, and metal ion). Nevertheless, it seems that the number of atoms in the QM region should be more than 100, and results with a smaller number of QM atoms should be considered with caution. The energy and structure of stationary points along reaction pathways are affected by the choice of functional and basis set used in the QM(DFT) method.72,73 Recently, it has been demonstrated that the calculated structure of TS depends on the simulation methods.74 Therefore, the method used should be suitable for a description of both carbohydrates and nucleophilic displacement reactions. Despite considerable progress in understanding of structure– function relationships of these enzymes, there are remaining challenges. Many glycosyltransferases exhibit conformational changes upon ligand binding, and the so-called ‘close’ and ‘open’ conformations were observed.30 The rate of these conformational changes often limits the overall rate of the enzymatic reactions. Recently, potent inhibitors of glycosyltransferases based on blocking the conformational change observed for these enzymes were developed.75,76 However, the character and role of these changes in the catalytic mechanism remains elusive and to get the complete picture of the reaction mechanism of GTs is one of the major challenges for the rational design of specific and potent inhibitors/ drugs for this class of enzymes. Without doubt, further developments in reliability and accuracy of QM(DFT)/MM methods will increase applications of these computational methods to study enzymatic reactions of carbohydrate processing enzymes. In silico experiments will more and more guide efforts in structure-based design of transition state analog inhibitors of glycoside hydrolases and glycosyltransferases. In general, the role of QM(DFT)/MM modeling methods in the area of glycoscience will unquestionably continue to grow. Acknowledgments This work was supported by Scientific Grant Agency of the Ministry of Education of Slovak Republic and Slovak Academy of Sciences (the project VEGA-02/0101/11), by the Slovak Academy of Sciences and National Science Council (Taiwan) bilateral project SAS-NSC JRP 2012/8, and the Research & Development Operational Programme funded by the ERDF (Centre of Excellence on Green Chemistry Methods and Processes, CEGreenI, Contract No. 26240120001, and Amplification of the Centre of Excellence on Green Chemistry Methods and Processes, CEGreenII, Contract No. 26240120025). References 1. Beyer, T. A.; Sadler, J. E.; Rearick, J. I.; Paulson, J. C.; Hill, R. L. Adv. Enzymol. 1981, 52, 23–175. 2. Schachter, H. Curr. Opin. Struct. Biol. 1991, 1, 755–765. 3. Kleene, R.; Berger, E. G. Biochim. Biophys. Acta 1993, 1154, 283–325. 4. New Comprehensive Biochemistry, Glycoproteins; Montreuil, J., Vliegenthart, J. F. G., Schachter, H., Eds.; Elsevier Science B.V.: Amsterdam, 1995; Vol. 29a.
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