Methyl transfer reactions catalyzed by cobalamin-dependent enzymes: Insight from molecular docking

Highlights

• Transfer of the methyl group in the catalytic cycle of MeTrs involves structural changes in their functional units.

• Methyl transfer in MetH and MtmBC most likely proceeds via a radical-based ET reaction rather than the S_N2 pathway.

• The changes in the structure of the methyl group acceptor incite a change in C-S bond distance.

• The changes in the structure of the Hcy domain facilitate the transfer of the methyl group to the substrate Hcy.

Abstract

Methyl transfer reactions, mediated by methyltransferases (MeTrs), such as methionine synthase (MetH) or monomethylamine: CoM (MtmBC), constitute one of the most important classes of vitamin B₁₂-dependent reactions. The challenge in exploring the catalytic function of MeTrs is related to their modular structure. From the crystallographic point of view, the structure of each subunit has been determined, but there is a lack of understanding of how each subunit interacts with each other. So far, theoretical studies of methyl group transfer were carried out for the structural models of the active site of each subunit. However, those studies do not include the effect of the enzymatic environment, which is crucial for a comprehensive understanding of enzyme-mediated methyl transfer reactions. Herein, to explore how two subunits interact with each other and how the methyl transfer reaction is catalyzed by MeTrs, molecular docking of the functional units of MetH and MtmBC was carried out. Along with the interactions of the functional units, the reaction coordinates, including the Co–C bond distance for methylation of cob(I)alamin (Co^ICbl) and the C–S bond distance in demethylation reaction of cob(III)alamin (Co^IIICbl), were considered. The functional groups should be arranged so that there is an appropriate distance to transfer a methyl group and present results indicate that steric interactions can limit the number of potential arrangements. This calls into question the possibility of S_N2-type mechanism previously proposed for MeTrs. Further, it leads to the conclusion that the methyl transfer reaction involves some spatial changes of modules suggesting an alternate radical-based pathway for MeTrs-mediated methyl transfer reactions. The calculations also showed that changes in torsion angles induce a change in reaction coordinates, namely Co–C and C–S bond distances, for the methylation and demethylation reactions catalyzed both by MetH and MtmBC.

Introduction

Cobalamins (Cbls, Fig. 1) are complex organometallic cofactors that catalyze a variety of enzymatic reactions [[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]]. The typical structure of Cbls consists of a corrin ring with a central cobalt (Co) ion which is equatorially connected to four nitrogens of the corrin macrocycle. In the base-on form, the metal center is axially ligated to the intramolecular 5,6-dimethylbenzimidazole base (DBI) at the lower axial position (α face) [1]. The upper axial position can be ligated with various ligands such as adenosyl (Ado), methyl (Me), cyano (CN), or hydroxy (OH⁻) [9,10]. In Cbls with upper and lower ligands, the oxidation state of Co is Co^III. Additionally, the Co can be found in two other oxidation states, including Co^II and Co^I [1,3]. Co^II and Co^I Cbls (Co^IICbl and Co^ICbl) appear as common intermediates in B₁₂-dependent enzymatic reactions [9,10]. Co^IICbl can be generated through the one-electron reduction of Co^IIICbl by a reducing agent or homolytic cleavage of the Co–C bond, while the heterolytic cleavage of the Co–C bond or the one-reduction of Co^IICbl generates Co^ICbl [1,4]. Changes in oxidation state also induce structural changes as well as the redox properties of Cbls [1]. From X-ray absorption spectroscopy it is apparent that Co^ICbl possesses a low-spin square-planner structure. However, upon binding with enzymes it can also appear as a penta-coordinated square pyramidal or hexa-coordinated octahedral geometry due to the formation of unusual Co^I…H interaction. These structural changes are induced by the change in the oxidation state of the Co atom during the catalytic cycle [[13], [14], [15]]. This change in the oxidation state, i.e. the reduction of Co^IICbl to Co^ICbl is arguably one of the most important aspects of B₁₂-dependent processes occurring in living organisms [9]. For example, in the family of Cbl-dependent methyltransferases (MeTrs), this process is used in the reactivation cycle of enzymes converting vitamin B₁₂ to biologically active forms such as MeCbl or AdoCbl [1].

MeCbl-dependent MeTrs are a class of enzymes which takes part in the biosynthesis of amino acids, carbon-dioxide conversion pathway as well as one-carbon metabolism of bacteria in many living organisms [5]. One of the most well-studied MeTrs is methionine synthase (MetH), which plays a pivotal role in catalyzing the biosynthesis of methionine [[6], [7], [8]]. In the first half of the catalytic cycle of MetH, the methyl group is transferred from methyl-tetrahydrofolate (CH₃H₄-Folate) to Co^ICbl (MeCbl) (Eq. (1)). In the second half of the reaction, MeCbl reacts with homocysteine (Hcy) to generate methionine by transferring the methyl group of MeCbl (Eq. (2)) [[9], [10], [11], [12]]. The displacement of the methyl group from the MeCbl cofactor, which is associated with the cleavage of the Co–C bond is the key step in this catalytic cycle. In the case of MeCbl-dependent MetH, this cleavage depending on the proposed mechanism is formally heterolytic or homolytic, whereas the cleavage of the Co–C bond in AdoCbl-dependent enzymes (AdoCbl = adenosylcobalamin) is homolytic.

CH₃H₄-Folate + Co^ICbl + H⁺ → H₄-Folate + MeCbl

MeCbl + homocysteine → Co^ICbl + methionine + H⁺

MetH is a modular enzyme which is consist of four different functional units [12]. MetH has four binding modules that are arranged linearly. Each module is connected by single interdomain linkers. The four modules of MetH include the methyl donor module (CH₃–H₄ Folate module), the methyl acceptor module (Hcy module), the Cbl binding module, and finally the adenosylmethionine (AdoMet) module. The methyl acceptor module is the N-terminal module and binds the CH₃–H₄ Folate. The methyl donor molecule binds Hcy, which is converted to methionine by the methyl transfer reaction. However, the mechanistic detail of the reaction involving demethylation of CH₃H₄-Folate and methylation of Hcy remains elusive from an experimental point of view, mainly because there is no structural data available regarding how two reacting modules of the protein interact during the enzymatic reaction. This is due to the lack of crystal structure of the reaction complexes.

Nevertheless, the mechanism of the activation of methyl donors such as MeCbl and CH₃–H₄ Folate has been extensively investigated using the techniques of computational chemistry [[6], [7], [8], [9], [10]], many mechanistic details associated with the catalytic cycle remain unclear. For example, S_N2 nucleophilic displacement has been suggested as one of the possible reaction pathways for methyl transfer reactions where the activation of substrate CH₃–H₄ Folate for transferring the CH₃ group is related to the protonation of N5 of CH₃–H₄ Folate [10,11]. The CH₃–H₄ Folate binding site of MetH consists of a pterin ring stabilized by hydrogen bonds formed by amino acids, such as asparagine and aspartic acid (Asp). In this mechanism, the N5 atom of –CH₃ donor (CH₃–H₄ Folate) is H-bonded with an acidic catalyst, particularly with the hydrophilic Asn₅₀₈ residue. It has been reported that this H-bonding interaction is involved in the stabilization of the protonation state. This protonation could take place either prior to or during the formation of the Co–C bond in the methyl transfer reaction [6,[16], [17], [18]]. However, alternative mechanisms, namely the single-electron transfer (SET) and oxidative addition (of CH₃-X group to Co atom in corrin ring) have also been proposed [19]. In the SET mechanism, the transfer of the methyl group from CH₃–H₄ Folate to Co^ICbl is completely reversible [19]. The three-step SET mechanism includes electron transfer, proton transfer as well as methyl group transfer. This mechanism allows the relaxation of requirments associated with the in-line geometry of the methyl group and donor moiety as well as the acceptor molecules [10]. Alternatively, the oxidative addition mechanism of –CH₃ includes proton uptake with simultaneous cleavage of the C–N5 bond. The major drawback of this mechanism is the formation of a three-centered bond between the N5–C bond of the CH₃–H₄ Folate and Co atoms Co^ICbl. The formation of this N5–C–Co bond is not possible inside the enzyme due to tremendous steric interaction in the protein backbone of two reacting modules, namely, the folate binding CH₃–H₄ Folate module and Cbl-binding modules of MetH [20]. Based on the stereochemistry of the transition state, it has been suggested that the oxidative addition mechanism occurs without any changes in the geometry of CH₃–H₄ Folate and Co^ICbl molecules [21]. To overcome these difficulties regarding the steric interaction inside the enzymatic environment, an alternative pathway, named reductive cleavage mechanism was proposed [22]. The major advantage of this mechanism is that it doesn’t require the formation of a three-centered bond and proximity between two reacting modules.

Moreover, one of the major drawbacks related to the proposed mechanisms of methyl transfer reactions catalyzed by MeCbl is the lack of structural data regarding the interaction of different modules, as well as the mechanism of activation and reformation of the Co–C bond. Due to the higher degree of conformational flexibility, the crystal structure of the whole enzyme (containing four modules of MetH) has not been solved yet. Therefore, from a structural point of view, it is not known how close two interacting modules can approach each other. This question is not specifically related only to MetH but also to other Cbl-dependent MeTrs, such as corrinoid iron-sulfur protein (CoFeSP) or Methanol: coenzyme M methyltransferase. In CoFeSP, it was expected that a 3–4 Å distance between two reacting centers of the methyl transfer process can facilitate the transfer of the methyl group through the S_N2 nucleophilic displacement. However, in reality, the two interacting modules of methyl transfer reaction inside CoFeSP couldn’t come closer than 8 Å due to steric interaction between protein backbones [5,10].

In this present study, we have aimed to provide computational insights into the mechanism of methyl group transfer catalyzed by MetH and monomethylamine:CoM (MtmBC) based on molecular docking of functional modules. MtmBC is a complex of monomethylamine methyltransferase (MtmB) and corrinoid protein MtmC, where the MtmB facilitates the transfer of methyl group of monomethylamine to the Cbl cofactor of MtmC. So far, theoretical studies of the mechanism of methyl transfer reaction involved a simplified model consisted of only isolated coring ring, CH₃–H₄-Folate, and Hcy domain [23,24]. Although a computational investigation of the enzyme-substrate (E-S) reaction complex has been conducted by docking the isolated CH₃–H₄-Folate substrate in the upper face of the Co^ICbl binding module of MetH, it did not consider the complete enzymatic environment and hence the possibility of a steric hindrance has been omitted [25]. The use of molecular docking with the overall structure of enzymatic modules in the theoretical modeling of the methyl transfer reaction to and from Co^ICbl is a new approach that will allow us to understand the effect of enzymatic environment and steric interaction on the mechanism of enzyme-mediated methyl transfer reaction.