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Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase domain of human Fatty Acid Synthase Pedro Paiva, Sergio F. Sousa, Maria João Ramos, and Pedro A. Fernandes ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00577 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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ACS Catalysis

Understanding the Catalytic Machinery and the Reaction Pathway of the Malonyl-Acetyl Transferase domain of human Fatty Acid Synthase Pedro Paiva, Sérgio F. Sousa, Maria J. Ramos and Pedro A. Fernandes*

UCIBIO@REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.

ABSTRACT

Human fatty acid synthase (hFAS) is a large multienzyme that catalyzes all steps of fatty acid synthesis, which is overexpressed in many cancer cells. Studies have shown that FAS inhibitors exhibit anti-tumor activity without relevant effects over normal cells. Therefore, the molecular description of active sites in hFAS should stimulate the development of inhibitors as anti-cancer drug candidates. The malonyl-acetyl transferase (MAT) domain is responsible for loading acetyl-CoA and malonyl-CoA substrates to the acyl-carrier protein (ACP) domain, a carrier for fatty acid reaction intermediates. In this work, we have applied computational QM/MM methods at the DLPNO-CCSD(T)/CBS:AMBER level of theory to study the MAT reaction mechanism. The results indicate that the initial catalytic stage occurs in two sequential steps: 1) nucleophilic attack on the thioester carbonyl group of the substrate through a concerted pathway that involves a Ser-His dyad; and 2) tetrahedral intermediate breakdown and release of the free coenzyme A. The Gibbs activation energies for the first and second steps are 13.0 and 6.4 kcal·mol-1, and 10.9 and 8.0 kcal·mol-1, whether the substrate transferred to the MAT domain is acetyl-CoA and malonyl-CoA, respectively. Both Met499 1 ACS Paragon Plus Environment

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and Leu582 form an oxyanion hole that lodges the negative charge of the substrate carbonyl, lowering the first step energetic barriers for both substrates. The mutation of the Arg606 residue by an alanine severely impairs the malonyl transfer reaction, while leading to a kinetic improvement of the transferase activity for acetyl-CoA, which is in agreement with earlier experimental studies. The results from this work encourage future studies that aim for the full comprehension of the MAT catalytic reaction and for the rational design of novel antineoplastic drugs that target this domain.

Keywords: fatty acid synthase, cancer, catalytic mechanism, transition state, QM/MM, ONIOM

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ACS Catalysis

INTRODUCTION In mammals, de novo fatty acid synthesis occurs mainly in the liver, following a conserved reaction pathway catalyzed by mammalian fatty acid synthase (mFAS)

1-3

. mFAS

is a multifunctional enzyme responsible for the biosynthesis of palmitic acid, a 16-C saturated fatty acid, using acetyl-CoA and malonyl-CoA as the initiation and elongation substrates, respectively 3-4. This cytosolic dimeric protein contains seven catalytic domains: ß-ketoacyl synthase (KS), malonyl/acetyl transferase (MAT), dehydrogenase (DH), enoyl reductase (ER), ß-ketoacyl reductase (KR), acyl-carrier protein (ACP) and thioesterase (TE)

1, 5

. The

sequential action of these catalytic domains leads to the production of fatty acids through a conserved set of cyclic reactions

1-2, 6

that share similarities with the polyketide synthetic

pathway performed by polyketide synthases (PKSs), a family of modular enzymes that produce a large set of natural bioactive compounds 7. In this cyclic pathway, an acetyl precursor is elongated by the sequential inclusion of two-carbon units derived from malonyl moieties. In most body tissues, the de novo biosynthesis of fatty acids occurs to a low extent. However, FAS gene is up-regulated in several human cancer cells and its overexpression is correlated with tumor malignancy 8-9. Studies have reported that high levels of the FAS gene are observable in many varieties of human cancers, including breast 15

colon , ovary

16

8, 10-11

, prostate

12-14

,

17

, and endometrium . Thus, FAS emerged as a very promising therapeutic

target in tumorigenesis and great efforts are being made in the research, development and enhancement of FAS inhibitors. The treatment of tumor cells with FAS inhibitors such as cerulenin (that irreversibly binds the KS domain) 9, orlistat (that irreversibly binds the active site of the TE domain, existing both as a hydrolyzed product and as a covalently bound intermediate)

18-19

, C75 (synthetic derivative of cerulenin that acts on KS, KR, ACP and TE

domains as a competitive irreversible inhibitor)

20-21

, Fasnall (a thiophenopyrimidine that

selectively targets FAS through competition with substrate intermediates over co-factor binding sites)

22

lead to fatty acid biosynthesis inhibition, induction of apoptosis and

inhibition of neoplasic cells growth 8-9, 23-24. As normal cells obtain their lipid supply from the dietary intake, these inhibitors do not compromise the survival of healthy cells in vitro and also do not constitute a source of toxicity in vivo

25-26

. This constitutes a serious advantage

when compared to other anticancer therapies that are currently employed and that usually lead to debilitating side effects

27-29

. The clinical relevance of inhibitors that target specific FAS

domains encourages new studies that aim at a better comprehension of each domain, their catalytic mechanisms, structures and contribution to the global biosynthetic reaction, 3 ACS Paragon Plus Environment

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providing not only mechanistic knowledge, but also atomic-level understanding to be employed in future rational drug discovery campaigns. Until this moment, no MAT-specific inhibitors have been developed, thus stimulating further studies of this domain and of its potential antitumor activity. MAT is a bifunctional domain of FAS composed of an α/β-hydrolase fold and a ferredoxin-like subdomain which, together, create a narrow and hydrophobic gorge that constitutes the active site

30-32

. This domain is responsible for the initiation of the fatty acid

biosynthesis pathway and is also involved in the subsequent elongation stages 2. First, it catalyzes the transfer of the acetyl moiety from acetyl-CoA onto the phosphopantetheine (PPT) group of the ACP domain, which then transfers this primer substrate to the KS domain. After that, MAT recharges the ACP domain with a malonyl moiety to be later incorporated in the growing fatty acid chain. While the mFAS MAT domain is able to recognize both the initiation and the elongation primer of the synthetic pathway, the loading of the primer (tipically acetyl or propionyl moiety) and chain-extender substrates in PKSs is catalyzed by specialized acyltransferases located in different modules 7. This means that different elongation primers (usually malonyl-CoA or methylmalonyl-CoA) may be utilized at each elongation step, which contributes to the diversity of polyketides that are produced by PKSs. It is proposed that the transfer reaction performed by mFAS and PKSs transferases occurs via a two-step ping-pong catalytic mechanism, in which a Ser-His catalytic dyad, that is positioned in a structural motif termed nucleophilic elbow, is crucial 7, 32-35. In the first step of the proposed mFAS mechanism, the conserved histidine (His683) enhances the catalytic serine’s (Ser581) nucleophilicity, favoring the attack of the labile thioester bond of the substrate and the subsequent formation of a tetrahedral intermediate. The negative charge of the intermediate is thought to be stabilized by an oxyanion hole nearby the carbonyl group. The intermediate is then resolved by the release of the acyl-enzyme complex and free CoA. In a second step, the free thiol group of the PPT arm of the ACP domain attacks the ester carbonyl, a tetrahedral intermediate is formed and resolved by the conserved histidine, through the re-protonation of the catalytic serine, leading to the release of the acyl-ACP product (Scheme 1).

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ACS Catalysis

Scheme 1. Schematic representation of the proposed mFAS MAT catalytic mechanism

7, 33-34

. R = acetyl or

malonyl moiety.

As previously mentioned, the MAT domain can recognize two distinct substrates, acetylCoA and malonyl-CoA, that act, respectively, as the initiation primer or chain-extender of the fatty acid synthetic pathway. According to Rangan and Smith

36

, this dual specificity of the

MAT domain is due to an arginine residue (Arg606) that is responsible for the binding of malonyl moieties to the transferase domain through ionic interactions with the carboxylate anion of the substrate. Additionally, through mutagenesis experiments, the same group demonstrated that the replacement of this arginine by an alanine residue severely compromises the transferase activity with the malonyl-CoA substrate and, on the other hand, dramatically increases the acetyl transferase activity. A recent study performed by Rittner et al. has also recognized R606 to be essential for the malonyl transfer reaction, as it successfully reproduced the compromised transfer of the malonyl moiety when the Arg606 was mutated by an alanine 32. The present work aimed to study the first stage of the catalytic mechanism of the MAT domain of FAS at a mechanistic atomic level, using quantum mechanics/molecular mechanics (QM/MM) simulation methods. Particularly, it examined how the acetyl and malonyl moieties are transferred from the CoA fraction to the human MAT domain and identified the contribution to this catalytic step of the residues that compose the oxyanion hole. Additionally, this work investigated the role played by Arg606 and the effects produced by its substitution for an alanine residue on the MAT mechanism. The results from this study provide new insights regarding transferases of the FAS/PKS families, the global fatty acid 5 ACS Paragon Plus Environment

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synthesis reaction and transition-state structures for the rational discovery and optimization of inhibitors with potential antineoplastic properties.

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ACS Catalysis

METHODS Model construction The human MAT-substrate model was carefully prepared from the crystal structure of the MAT domain of human FAS (PDB ID 2JFK, 2.4 Å resolution)

30

, which was

subsequently subjected to minor modelling, as no human MAT-substrate complex is currently known. The human FAS KS-MAT di-domain structure (PDB ID 3HHD)

37

was also taken

under consideration during the design of the MAT model. However, as Pappenberger and colleagues mentioned in their work, the crystal exhibits MAT in two closed conformations that do not readily allow the entry of a larger molecule, such as malonyl-CoA or acetyl-CoA substrates, being that the motive why PDB ID 2JFK was selected to pursue the study. This structure consists of an asymmetric unit with four molecules, each one complexed with CoA and malonylcysteine ligands that are structurally identical and active as monomeric enzymes. Consequently, only one chain was kept in the model (chain D). Given that the CoA ligand is positioned just outside the active site pocket and that the malonylcysteine ligand adopts a non-optimal positioning for catalysis (the carbonyl carbon is located at more than 5 Å from the catalytic Ser581 hydroxyl group), both were removed from this structure. The superimposition of chain D with E.coli FabD (the bacterial homologue of MAT) complexed with malonyl-CoA (PDB ID 2G2Z, 2.8 Å resolution) 38 showed a similar steric orientation of the active site residues. This specific complex was obtained through a soaking experiment, immediately after the transferase reaction took place, thus indicating that the active site residues and substrate should adopt a position that is very close to the catalytically active one. PDBeFold analysis

39

revealed 22% sequence identity between human MAT and bacterial

FabD, as well as a RMSd value of 1.99 Å regarding C-α atoms of matched residues. The comparison of all amino acid residues closest to the substrate (< 6 Å) revealed a sequence identity of 58% (7/12). Furthermore, since the malonyl moiety of the processed substrate is covalently bound to the catalytic serine in FabD complex and given the similarities between the human and bacterial transferase active sites, this moiety was also at a nearly adequate distance for the reaction to occur in the human domain. Therefore, the FabD malonyl moiety was transferred to the MAT model, keeping the coordinates obtained from the previous structural alignment. Visual inspection of the substrate-binding pocket showed that the side chain of Met499 was blocking the channel leading to the active site of the human MAT model. The coordinates of this amino acid were modelled using the homologous porcine FAS (PDB ID 2VZ8, 3.22 Å resolution) 40 Met499 as an open channel template. Hydrogen atoms, TIP3P water molecules

41

and Na+ counter ions were added to the 7

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system using the XLEaP module of the AMBER 12 package

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42

. Standard protonation states

for all amino acids at pH 7.0 were adopted and carefully checked. H++ web-server software 43 was used to validate the protonation state of titratable residues chosen by XLEaP. Both methods only differed in the protonation state of three histidine residues, which were considered charged by H++ server and neutral by XLEaP. One of the His was His683, which is reported to be involved in the chemical reaction and has to be neutral and protonated at the δ nitrogen for the mechanism to be feasible 7. The other two His (His614 and His751) are exposed to the solvent, far from the active site. Given their positions, their influence in the reaction mechanism is expected to be negligible. Even though, we decided to keep them neutral given their exposure to the solvent (pH of 7.5) and the pKa of His in water (6.9). In all other His residues, a proton was added to the ε nitrogen atom of the imidazole ring. To alleviate unfavorable interactions and possible steric clashes, an energy minimization was performed using the SANDER module of the AMBER 12 package

42

. Previously to the

minimization and molecular dynamics (MD) protocol, it was necessary to parametrize the ligand, malonyl-CoA. Since the visual inspection of the substrate in the context of the enzyme revealed that the CoA moiety was positioned along the gorge and extended towards the outside of the substrate-binding pocket, the non-interacting portion of the CoA was truncated, as it should not play a crucial role in the catalytic reaction. Thus, to describe bonds, angles, dihedrals and van der Waals radii for the truncated substrate, the standard ANTECHAMBER procedure based on the General AMBER Force Field (GAFF)

44

was employed. RESP

(restrained electrostatic potential) fitting method was employed to derive atomic charges at the HF/6-31G(d) level of theory with the Gaussian 09 software (version D)

45

. The

minimization protocol encompassed five consecutive steps in which the constraints exerted on the system were gradually removed. In the first step, only the truncated CoA moiety was minimized; in the second one, only water molecules were minimized; in the third one, only hydrogen atoms were allowed to move freely; in the fourth step, the side chains were minimized and, in the fifth and final step, no constraints were applied to the system. After the minimization routine, the system was submitted to a classical MD simulation. The SHAKE algorithm 46 was used through the MD procedure to constrain all bonds comprising hydrogen atoms. MD started with a 50 ps equilibration phase, in which the system was progressively heated from 0 to 310 K. This allowed the temperature of the system to increase, while the density remained constant. The equilibration phase was followed by a short 0.5 ns run in the NPT ensemble at 310 K and 1 bar, in which a Langevin thermostat and a Berendsen barostat were used. Such a short run was used to relax the system due to the modelling but without moving it from the folding minimum captured in the X-ray experiment. A root-mean-square 8 ACS Paragon Plus Environment

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ACS Catalysis

deviation (RMSd) analysis of both the protein and active site residues was performed to access the stability of the system during the MD course (supporting information, Figure S1). Catalytic-relevant bond lengths were also followed throughout the simulation time. Interatomic distances inferior to 3.5 Å between Oγ(Ser581)-Ccarbonyl(malonyl-CoA) and under 3.0 Å between Hγ(Ser581)-Nε2(His683), the presence of ionic interactions between the carboxylate group of malonyl-CoA and Arg606, and the positioning of the substrate’s oxyanion towards two backbone amines were used as criteria to select structures as candidates for the final MAT-substrate model. About 11% of the complexes gathered from the MD protocol satisfied the selection criteria and the structure that exhibited closer Oγ-Ccarbonyl and Hγ-Nε2 distances, as well as stronger carboxylate-Arg606 interactions, was elected to pursue the QM/MM calculations. This structure was aligned with the malonyl-loaded MAT domain of the recently published murine KS-MAT di-domain crystal (PDB ID 5MY0, chain D) 32 and both active sites were compared (supporting information, Figure S2). Catalytic-relevant interactions are conserved among the two models, particularly those established between Ser581 and His683, between Ser581’s Oγ and the substrate’s carbonyl carbon, as well as the ionic interactions performed between the carboxylate anion of the malonyl moiety and the guanidinium group of Arg606. Naturally, we remain aware that any slight imprecision performed during the modelation protocol may produce an impact on the QM/MM results that were based on the modelled structure. Particularly, we think that the prediction of the binding pose of the substrate is a sensitive aspect of the modelling, as the substrate’s moiety was transferred from another system (FabD, PDB ID 2G2Z). Fortunately, knowing that reactive substrates form fundamental interactions with the catalytic active site residues, the prediction of the binding pose of a reactive substrate is easier and more accurate than predicting the binding position of non-reactive ligands. We find difficult to picture another binding pose that would still be catalytically competent as the one observed in the MD structure. In this sense, we are confident that our model should be able to successfully reproduce the structural and catalytic phenomena characteristic of the MAT domain.

QM/MM: To explore the potential energy surface of the MAT-substrate model, ONIOM QM/MM calculations were performed with the Gaussian 09 (version D) software. Other computational techniques, such as the cluster model or QM/MM molecular dynamics approaches, have also been successfully employed for the study of catalytic mechanisms 47-50. ONIOM QM/MM has been widely used in enzymatic studies

50-53

, treats the most relevant 9

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atoms of the active site with a high level of theory, while the remaining portion of the enzyme is at the molecular mechanics level. The ONIOM subtractive scheme embedding was employed and the GaussView 5.0 software

55

54

with electrostatic

was used to define two layers

that would be posteriorly treated with different levels of theory. The QM (high level) layer included atoms that were thought to participate, directly or indirectly, in the MAT reaction, while the MM (low level) region included all the remaining atoms of the system. Thus, the QM region (Figure 1) consisted of the full Ser581, the side chain of His683 and Arg606, a portion of the backbone of Gly498, Met499 and Leu582, and the malonyl moiety plus the sulphur atom and the two adjacent CH2 groups of the truncated malonyl-CoA substrate (total of 59 atoms), while the MM region accounted for the remaining 6311 atoms.

Figure 1. Three-dimensional structure of the MAT-malonyl-CoA reactant complex (6370 atoms). Right: closeup of the QM region, with malonyl-CoA as substrate.

The high-level layer was treated using density functional theory (DFT) at the B3LYP/631G(d) level of theory for geometry optimizations and linear transit scans, while the MM layer was treated at the molecular mechanics level, described with parm99SB parameters. Hydrogen atoms were used as link atoms

56

to complete the valences of the bonds that cross

the QM/MM frontier. As MAT catalyzes the transfer of two different moieties (malonyl and acetyl) in an identical reaction, a QM/MM model of MAT-acetyl-CoA was also built. The substrate of the MAT-malonyl-CoA structure obtained from the MD was modelled through the replacement of the terminal carboxylate and the adjacent CH by a methyl group, 10 ACS Paragon Plus Environment

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ACS Catalysis

generating the acetyl moiety. The R606A mutant models were built using the MAT(WT)acyl-CoA complexes, in which the side chain of the Arg606 was replaced by a methyl group. The mutated models were submitted to sequential AMBER (MM) and ONIOM (QM/MM) optimizations, so that the system was allowed to adapt to the perturbation introduced by the mutation. Thus, depending on the model being studied, the full system consisted of 63546370 atoms. For the four optimized models (MAT(WT)-malonyl-CoA, MAT(WT)-acetylCoA, MAT(R606A)-malonyl-CoA and MAT(R606A)-acetyl-CoA), a potential energy profile was obtained as a result of linear transit scan calculations, using the distance between the oxygen of the Ser581 hydroxyl group and the carbonyl carbon of the substrate (either malonyl-CoA or acetyl-CoA) as a reaction coordinate for the first step. Regarding the second step of the hypothesized catalytic mechanism, the distance between the sulphur atom of the free CoA molecule and the hydrogen previously transferred to His683 was established as the reaction coordinate. These distances were shortened by 0.05 Å in each consecutive step, while the rest of the system was optimized for every reaction coordinate value. The minima and maximum of the potential energy surface (PES) were used as guessing structures for the subsequent free optimization of the reactants, products and the transition-state. Internal reaction coordinate (IRC) calculations were also performed to confirm the validity of the minima obtained. Vibrational calculations at 0 ºC were carried out for each optimized structure to confirm their status as minima (zero imaginary frequencies) or transition-state (one imaginary frequency) and to determine entropic and thermal corrections, within the RRHO formalism. Afterwards, single-point QM/MM energy calculations were performed at the B3LYP/6-311+G(2d,2p) and including the Grimme’s D3 corrections. To improve the accuracy of the results to the coupled cluster level, the recently developed domain-based local pair natural orbital coupled cluster method with single, double, and perturbative triple excitations (DLPNO-CCSD(T))

57-58

was employed. This method, that already found

applications among diverse subjects of chemistry and biochemistry

59

, has shown to be

capable of delivering results that closely resemble those obtained with the canonical method, at a significant lower computational cost 60-61. In this context, the QM regions of all stationary points were submitted to DLPNO-CCSD(T) single-point energy calculations with the ORCA (version 4.0.1) software

62

, using the cc-pVDZ and cc-PVTZ basis sets, as well as the cc-

pVDZ/C and cc-PVTZ/C correlation fitting basis sets (in ORCA nomenclature), keeping the cutoff parameters of DLPNO at their default values. Truhlar’s extrapolation method

63

was

employed for the extrapolation to the complete basis set (CBS) limit through the combination of the DLPNO-CCSD(T) energies obtained with cc-pVDZ and cc-pVTZ basis sets. Note that the lack of diffuse functions in the two used basis sets is not reflected in the final energy 11 ACS Paragon Plus Environment

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values because they are used to obtain an extrapolation to the CBS limit, where all basis sets insufficiencies basically disappear. The DLPNO-CCSD(T)/CBS method provided energies that differ, in average, 2.1 kcal.mol-1 of those obtained at the DFT (B3LYP/6-311+G(2d, 2p)D3) level. We are aware that this particular extrapolation approach presents two minor limitations: i) the use of two short basis sets for the extrapolation (particularly cc-pVDZ) and ii) the fact that it was developed for the extrapolation from canonical CCSD(T) energies and not from those obtained with the DLPNO approximation. As previously mentioned, recent studies have demonstrated that the DLPNO-CCSD(T) energies are very close to those obtained with the canonical coupled cluster method

61

, which suggests that the DLPNO-

CCSD(T) extrapolated energies should also be very similar to the canonical extrapolated ones. On the other hand, as it was reported that a triple-ζ basis set brings DLPNO-CCSD(T) sufficiently close to its basis set limit and that DLPNO-CCSD(T) with a double-ζ basis set is more accurate than many DFT functionals 60, we strongly believe that the extrapolation from cc-pVDZ and cc-pVTZ should provide sufficiently accurate energies that are close to the complete basis set limit. It is a fact that augmented basis sets should be preferred for the CCSD(T) CBS extrapolation, given their higher proximity to the basis set limit 64. Taking that in consideration, DLPNO-CCSD(T) calculations with the aug-cc-pVTZ basis set were also performed. However, given the substantial increase in the computational cost and the very small energetic difference (≤ 0.6 kcal.mol-1, in average 0.3 kcal.mol-1) when compared to the DLPNO-CCSD(T)/CBS level, the latter was elected for future calculations. Table S1 (supporting information) provides a comparison between the energies obtained at the DFT (B3LYP/6-311+G(2d, 2p)-D3), DLPNO-CCSD(T)/CBS and DLPNO-CCSD(T)/aug-ccpVTZ levels. The ORCA software was also used to carry out single-point calculations of the QM regions using DFT at the B3LYP/6-311+G(2d,2p)-D3 level. The difference between the DLPNO-CCSD(T)/CBS and the B3LYP/6-311+G(2d,2p)-D3 single-point energies of each QM region was added to each QM/MM B3LYP/6-311+G(2d, 2p)-D3:AMBER single-point energy previously calculated with the Gaussian 09 software. This means that the polarization of the QM region by the MM point charges, and the interaction between the QM electron density and the MM point charges is calculated at the B3LYP/6-311+G(2d, 2p)-D3 level within the electrostatic embedding formalism. These corrected energies, together with the entropic and enthalpic corrections obtained from the vibrational calculations at 0 ºC, allowed the calculation of the final Gibbs activation and reaction free energies of the catalytic steps in study.

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ACS Catalysis

RESULTS AND DISCUSSION QM/MM calculations were performed in the assembled model to clarify the catalytic mechanism for the transfer of the acyl moiety from the CoA to MAT. The results for the wildtype (WT) enzyme will be presented first, followed by the results of the calculations made to elucidate and quantify the contribution of the oxyanion hole for the first reaction stage. Finally, the results obtained with the Arg606 mutant (R606A) MAT will be outlined.

Figure 2.

Free-energy profile for the acyl transfer reaction from CoA to MAT at the DLPNO-

CCSD(T)/CBS:AMBER level of theory.

Wild-type MAT Step 1 – Nucleophilic attack on the thioester carbonyl group Previous studies have suggested that the acyl transfer mechanism between CoA and MAT initiates with the deprotonation of the active site serine by a neighboring histidine residue (the catalytic dyad) 7. This would enhance the nucleophilicity of the serine and facilitate the attack of the serine on the carbonyl carbon of the substrate. The computational calculations revealed that if the deprotonation of Ser581 took place, its Oγ atom deprotonated the nearby Arg606 in a combined process with an energetic barrier of 21 kcal·mol-1, clearly exceeding the 14.7-15.6 and 14.6-15.8 kcal·mol-1 barriers determined experimentally for the MAT-acetyl-CoA and MAT-malonyl-CoA complexes

32, 36

. Furthermore, no Ser581-Oγ-

intermediate (ready to attack the carbonyl carbon) was detected, which led to the rejection of 13 ACS Paragon Plus Environment

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this two-step pathway. These findings inspired the proposal of a mechanism that starts with a concerted reaction involving the two steps previously mentioned (Scheme 1). Indeed, the results demonstrated that the Ser581 deprotonation by His683 and the nucleophilic attack on the thioester carbonyl group of the substrate can take place through a concerted step (step 1) with free-energy barriers of 13.0 and 10.9 kcal·mol-1 (reaction free energies of 3.6 and 4.8 kcal·mol-1) for acetyl-CoA and malonyl-CoA substrates, respectively (Figure 2). These values are slightly lower than the energetic barriers obtained experimentally by Rangan and Smith 36, which suggests that this concerted step (step 1) is not the rate-limiting step of the catalysis. As the attacking Oγ of Ser581 approaches the thioester carbonyl carbon of the substrate, the HγOγ bond of Ser581 breaks down and the proton moves towards His683. These Ser581-His683 interactions are a reminiscent of analogous interactions observed in enzymes that contain a Ser-His-Asp catalytic triad, such as those of the α/β-hydrolase

65

66-67

, serine protease

and

68

subtilisin-like enzyme families . Moreover, acyltransferases including 6-deoxyerythronolide B synthase from PKS (DEBS) 7, E.coli malonyl-CoA-ACP transacylase FabD mitochondrial methyltransferase transacylase

69

30

38

, human

and H.pylori malonyl-CoA acyl carrier protein

constitute examples of MAT-like biological systems whose catalytic

mechanism is also centered on a Ser-His