Theoretical Evaluation of the Reaction Mechanism of Serine

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Theoretical Evaluation of the Reaction Mechanism of Serine Hydroxymethyltransferase Jirapat Santatiwongchai, Duangkamol Gleeson, and Matthew Paul Gleeson J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b10196 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 11, 2018

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The Journal of Physical Chemistry

Theoretical Evaluation of the Reaction Mechanism of Serine Hydroxymethyltransferase Jirapat Santatiwongchai,1 Duangkamol Gleeson2 and M .Paul Gleeson1,3* 1 2

Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

Department of Chemistry, Faculty of Science, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand. 3

Department of Biomedical Engineering, Faculty of Engineering, King Mongkut's Institute of Technology Ladkrabang, Bangkok 10520, Thailand.

To whom correspondence should be addressed. Phone+ :66-8-69779678 .Fax+ :66-2-3298346 AUTHOR EMAIL ADDRESS :[email protected]

1

ACS Paragon Plus Environment

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ABSTRACT Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate (THF) to glycine and 5,10‐methylene THF. SHMT is a folate pathway enzyme and is therefore of considerable medical interest due to its role as an important intervention point for anti‐malarial, anti‐cancer and anti‐bacterial treatments. Despite considerable experimental effort the precise reaction mechanism of SHMT remains unclear. In this study we explore the mechanism of SHMT to determine the roles of active site residues and the nature and the sequence of chemical steps. Molecular dynamics (MD) methods were employed to generate a suitable starting structure which then underwent analysis using hybrid quantum mechanical/molecular mechanical (QM/MM) simulations. The QM region consisted of 12 key residues, two substrates and explicit solvent. Our results show that the catalytic reaction proceeds according to retro‐aldol synthetic process with His129 acting as the general base in the reaction. This rate determining step involves the cleavage of the PLP‐serine aldimine C‐C bond and the formation of formaldehyde in line with experimental evidence. The pyridyl ring of the PLP‐serine aldimine substrate exists in deprotonated form, being stabilized directly by Asp208 via a strong H‐bond, as well as through interactions with Arg371, Lys237 and His211, and with the surrounding protein which was included electrostatically. This knowledge has the potential to impact the design and development of new inhibitors.

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1.0 Introduction Malaria is a major public health issue worldwide, particularly in tropical and subtropical countries where almost half of the world’s population reside. It was reported in 2017 that over 200 million people worldwide are infected with this parasitic disease, approximately 450 thousand of whom will die.1 Despite the existence of numerous antimalarial drugs, the treatment of malaria continues to be a major challenge due to disease resistance.2‐5 Efforts to tackle this endemic problem involve the use of combination therapies, however, there is a clear understanding that new chemical classes acting at known targets, or new drugs that act at novel targets are urgently needed.6‐7 This has prompted renewed effort by non‐ governmental organizations and academic groups to work in collaboration with the pharmaceutical industry to develop more effective strategies and treatments.8‐10 The folate pathway plays an essential role in one‐carbon metabolic biosyntheses of methionine, purines and pyrimidines needed for DNA reproduction and cell growth.11‐12 Cells that are required to undergo rapid division are very sensitive to the inhibitors of the folate pathway and therefore make good targets for medicines. Folate inhibitors have therefore emerged as effective agents to treat cancer, bacteria and parasitic infections.13 Three clinically validated folate targets exist at present; dihydropteroate synthase (DHPS), dihydrofolate reductase (DHFR) and thymidylate synthase (TS).14‐16 While the combination therapy of sulfadoxine (DHPS inhibitor) and pyrimethamine (DHFR inhibitor) was a mainstay in the treatment of malaria, it is now employed to treat only uncomplicated malaria infections.17 Current front line malaria treatments typically involve artemisinin‐based combination therapies.18 which are themselves are beginning to face drug resistance issues.19‐20 Efforts to develop new DHFR inhibitors21‐23 and as inhibitors for other folate targets are currently underway.15, 21 Serine hydroxymethyltransferase (SHMT) is a folate pathway enzyme located one step after DHFR and has received considerable attention from the medicinal chemistry and structural biology community.24‐29 SHMT is a pyridoxal‐5’‐phosphate (PLP)‐dependent enzyme, that catalyzes the conversion of L‐serine and tetrahydrofolate (THF) to glycine and 5, 10‐methylenetetrahydrofolate (5,10‐MTHF). SHMT is a highly versatile enzyme that can also catalyze THF‐independent aldolytic cleavage, decarboxylation, racemization, and transamination reactions.30 This versatile enzyme has evolved to stabilize a diverse set of intermediates and transition states. Experimental data and QM calculations point to the rate determining step in the formation of 5,10‐MTHF as being the breaking of the C‐C bond of the external‐ serine aldimine substrate (Scheme 1).31‐32 The enzyme exists in homodimeric or homotetrameric form, the

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former being the minimum necessary for catalytic function. The active site is located at the interface of two individual monomers and is surrounded by five conserved loops from the first chain and the second chain (Figure 1).33 The active site requires two substrates as shown in Scheme 1, a PLP aldimine and tetrahydrofolate (THF). The PLP aldimine can exist in two forms within SHMT, bonded to Lysine as an internal aldimine, and to a serine residue in the so called external aldimine form or PLP‐serine aldimine. The latter is believed to be involved in the rate determining chemical transform step.31, 34 PO4

2-

H2C N

N

OH

H N

OH

CH

OH

+

O

PLP-serine aldimine

R

NH

N H

N

NH2

PO4

H

NH O

H N

2-

N N

OH

CH

OH

+

N

NH

N

O

N CH2

NH2 H2O

O

R

tetrahydrofolate (THF)

PLP-glycine aldimine 5,10-methylenetetrahydrofolate

Scheme 1 SHMT proposed mechanism based on retro‐aldol reaction. Shown in the enolimine form.

Figure 1 Model of SHMT showing active site region at the interface of the dimeric protein structure.

THF is bound alongside PLP in the SHMT active site as illustrated in Figure 2.33 The para‐ aminobenzoic acid (pABA) portion of THF binds within the active site, making interactions with Tyr63 (π‐π stacking), Phe134 and Phe266 (edge‐to‐face). Interactions are made between the pterin ring of THF and active site bound water molecules which themselves connect to Asn356. PLP is surrounded by a network of H‐bond interactions with Arg371, Lys237, His211, Thr183 and Asp208 (Figure 2).35 Asp208 H‐bonds to the pyridyl nitrogen of PLP and is considered important for the stabilization of PLP during the catalytic 4


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reaction. The protonation state of PLP within PLP‐dependent proteins shows a marked dependence on the specific residue adjacent to the pyridyl nitrogen as well as other aspects of the active site environment. The pyridyl nitrogen can exist in deprotonated36‐37 and protonated forms38 depending on residue adjacent to the pyridyl nitrogen amongst other factors.39 Molecular dynamics (MD) simulations on SHMT suggest it exists in the protonated form.40 PLP can also exist in either a keto or enol form (Figure 3). The relative preference for the keto or enol form is known to depend on its environment.32, 40‐42 Theoretical studies on SHMT suggest PLP exists in the keto form based on MD40 and gas‐phase QM calculations.32 This is in line with suggestions from more extensive studies on related PLP dependent protein aspartate aminotransferase.41 Nevertheless, hybrid quantum mechanics/molecular mechanics (QM/MM) studies of other PLP dependent proteins such as alanine racemase and L‐DOPA decarboxylase42 suggest that the enol PLP tautomers can be observed.41 Interestingly, neutron structures of the PLP dependent protein aspartate aminotransferase indicated that local active site effects, as well as the protonation state of the pyridyl nitrogen, can significantly affect the relative stability of the two tautomers.38

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Figure 2 2D (left) and 3D representations of the SHMT active site model employed in this study. H‐bonds and ‐ stacking interactions are illustrated with dashed red and grey lines respectively. The N5‐C bond formed during the reaction is indicated with a blue dashed line.

Figure 3 Potential protonation states on PLP aldimine structure. Two tautomers which a proton protonated on the phenolic oxygen (a) enolimine and imine nitrogen (b) ketoenamine. (c) pyridyl nitrogen in deprotonated form and (d) pyridyl nitrogen is protonated.

Two catalytic mechanisms for SHMT have been put forward for SHMT involving (a) retro‐aldol cleavage30, 32, 35, 43‐44 and (b) direct nucleophilic attack of PLP by THF.30, 43, 45 The retro‐aldol mechanism sees the cleavage of PLP‐serine C‐C bond followed by the condensation of formaldehyde with THF. This is the most commonly accepted proposal (Scheme 2). The direct displacement of the Cα atom of PLP‐serine

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aldimine has been proposed to occur via nucleophilic attack by the N5 of THF. QM calculations of the two mechanisms indicate that the enzyme preferentially follows a retro‐aldol process.32 Two primary candidates for the role of general base in the SHMT catalytic reaction His129 are Glu56.30, 32, 43 Structural analysis of Threonine aldolase, a structurally related PLP‐dependent enzyme, which follows a very similar retro‐aldol process involving acetaldehyde46 is reported to utilize histidine as the general base.47 Glu56 was an early candidate for general base, however more recent experimental data has indicated that it is not involved in the proton abstraction.44, 48‐49 Mutational analysis of Glu75 in rcSHMT and Glu74 in scSHMT (Glu56 in PvSHMT) suggested that Glu56 is instead involved in the condensation step and not the retro‐aldol cleavage step.49 Further structural studies suggested that Glu56 could serve as the acid catalyst in the later steps but not for the retro‐aldol mechanism,44 that it is a critical residue in binding SHMT substrates and can exist in both protonated30, 35 and deprotonated forms.27 Hybrid QM/MM methods have been used extensively to probe the sequence of event of many enzymatic processes. In these calculations the protein molecule is treated using two different levels of theory.50‐56 QM/MM methods have been used recently to study other critical enzymes on the folate pathway57‐60 as well as other PLP‐dependent enzymes.36, 41 However, to date only rather limited QM studies on small gasphase calculations on SHMT have been performed but have not considered many of the unanswered questions regarding the reaction mechanism.32 In this study an ONIOM‐based61‐62 electrical embedding methodology has been used to investigate aspects of the SHMT catalytic mechanism. The QM/MM model has been used to (a) identify the general acid/base involved in the SHMT catalytic reaction, (b) the most probable protonation states of the active site residues, and (c) the lowest energy pathway associated with the formation of products. A clearer understanding of the catalytic process that occurs within SHMT will have significant implications for future structure based drug design applications associated with SHMT.

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tetrahydrofolate (THF)

PLP-serine aldimine Asp208-H

PO4

N

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R

2-

NH HN

HO Arg 371 H2N

+ NH2

N O

O

O

CH2 O H

N N H

–

PO4

NH

NH H

HN

O N

N N H

–

O

PO4

NH

NH

O

H-Base O

NH +

H O

N O

N N H

–

O

NH

NH

H2O

N O

H2C N O

–

NH

2-

R

NH N

N H

O

PO4

NH2

R

2-

+

HO

NH

H

O

PO4

NH2

R

2-

N HO

NH2

R

2-

H

HO

NH

Base:

NH2

+

NH H2C

HO

H2O

N O

N

NH

O

–

O

N N H

NH2

Scheme 2 The retro‐aldol reaction mechanism proposed for SHMT.30, 32, 35, 43‐44

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2.0 Methods A crystal structure of SHMT was downloaded from the RCSB protein databank and prepared as follows (PDB ID: 4OYT, resolution of 2.4 Å,33). All amino acids in chain C of the crystal structure were removed since the catalytic mechanisms requires a tight dimer active site pocket which is located between chain A and chain B. The protein was prepared for QM/MM by performing restrained MD on 5‐ hydroxymethyl‐THF and PLP‐glycine aldimine bound to the active site. To this end the N5‐formyl group of the THF analog present in the X‐ray structure was modified to give the corresponding alcohol and D‐serine was converted to glycine (Figure S3). The pyridyl nitrogen of PLP‐serine‐aldmine was modelled in the protonated form. The protonation state of the amino acid residues were defined based on their predicted pKa calculated using Propka3.163‐64 and a visual assessment of the local interactions. Histidine residues were defined as HID except for His274 on chain A and His129, His132, His211 and His326 of the chain B, which were defined as HIE. His236 was modelled as HIP due to the possibility of interacting with the phosphate group of PLP substrate. 2.1 Molecular Dynamics Simulations The dimeric SHMT complex underwent a short equilibration procedure to produce a suitable starting structure for QM/MM. Molecular mechanics optimization and dynamics (MD) simulations were performed in Gromacs program v4.5.465 using the AMBER99SB forcefield.66‐67 The restrained electrostatic potential (RESP) charge of the ligands were determined from a Hartree‐Fock calculation with the 6‐31G(d) basis set. Ligands topology files were created using the ACPYPE script with the GAFF forcefield.68‐69 The complex was solvated in a cubic box of TIP3P water70‐71 with a minimum distance of 12 Å between the protein and box edge. An NaCl ionic strength (0.15 M) was used to neutralize the system. Prior to molecular dynamics (MD) simulations, the system was minimized to an RMS gradient of 10 kJ mol‐1 nm‐1 to relax any steric conflicts. The system was then heated to 300K and equilibrated under the NVT ensemble for 500 ps, followed by 1 ns of MD simulations under NPT scheme with the Parrinello‐Rahman barostat72 at 1 atm and a restraint protein backbone (force constant 1000 kJ/mol nm2) to maintain their active site in its active conformation. Simulations utilized a step of 0.001 ps, in conjunction with the LINCS algorithm, a 2 fs time step and particle mesh Ewald.73 The structure obtained from the final step of MD was then minimized to an RMS gradient of 10 kJ mol‐1 nm‐1. The 5‐hydroxymethyl‐THF and PLP‐glycine aldimine substrates were then used to generate the THF and L‐serine substrate coordinates used in the QM/MM analysis.

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2.2 QM/MM Calculations QM/MM calculations were performed using the Gaussian 09 program. The MD derived protein structure was prepared for by removing solvent beyond 16 Å of the active site residues. Amino acid residues beyond 25 Å from the active site were also removed for computational considerations. The sidechains of charged amino acids excluded were replaced with Na+ or Cl‐ atoms respectively to preserve the unique electrostatic effects of the protein dimer. 52 positively charged amino acids and 59 negatively charged amino acids were therefore added. The QM subsystem consisted of the key amino acid sidechain and backbone atoms that are reported to play a direct role in ligand binding or catalysis. The QM region includes the two substrates, tetrahydrofolate (THF) and PLP serine aldimine, the side chains of Tyr63, Gly128, His129, Phe134, Thr183, Asp208, His211, Lys237, Phe266, Arg371 and six water molecules. The interactions present in QM region are illustrated in Figure 2. Additional 3D illustrations of the active site be found in Figure S1 and S2. Atoms within 10 Å of active site were treated flexibly during the QM/MM calculations, apart from those that crossed the QM/MM interface. The QM region consisted of 198 atoms while the MM region consisted of 9,259 atoms. QM/MM calculations were performed in Gaussian 09 using the ONIOM electronic embedding scheme.74 All structures were fully optimized using the M062x functional, developed by Truhlar and co‐workers,75‐76 and the 6‐31G(d) basis set. The MM region was treated using the AMBER forcefield.69 Minima were confirmed as having no imaginary frequencies and transitions states a single major imaginary frequency corresponding to the expected bond breaking/forming process under investigation. The free energies were calculated at M062x‐6‐31G(d) method. A further correction in the form of single‐point energies at 6‐31+G(d,p) level (∆Esp) were also performed.

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3.0 Results & Discussion Molecular dynamics (MD) was employed to generate a suitable starting structure for the QM/MM study. The protein backbone was restrained to maintain a structure in what was already an ideal catalytically relevant conformation. Both substrates were bound in conformations suitable to undergo reaction negating the need to explore additional protein‐ligand conformational space. We therefore restrained the protein backbone but allowed the substrates, amino acid sidechains and water molecules to move freely. The system was equilibrated for 1 ns of MD followed by minimization to produce a low strain, protein in an experimentally relevant, catalytically active conformation suitable for further analysis. The Cα atom RMSD between the structure obtained from MD and the X‐ray structure was found to be 0.298 Å showing the structure remained in its catalytically relevant conformation (Figure S4, Figure S5). The Cα‐Cβ distance between the methanol carbon and the C of PLP‐glycine aldimine was 4.9 Å on average over the course of the MD simulations indicating the substrates remained in a configuration capable of reacting (Figure S6). THF was shown to display greater flexibility in the SHMT active site pocket, primarily as a result of the solvent exposed carboxylate groups (Figure S7). This structure was subsequently used to investigate the protonation states of PLP and adjacent residues, the identity of the general base in the reaction, and the relative stability of different intermediate, products and transition states needed to connect the reactant to product. The structures of the ketoenamine and enolimine stationary points obtained using the QM/MM model are illustrated in Figure 4 and Figure 5 respectively. Additional geometric parameters for the enolimine based reaction mechanism are provided in Table 1 and Mulliken charge distributions per substrate/residue in Table S1. We have compared the QM/MM energies at the M062x/6‐31G(d)//AMBER level to single point energies at the M062x/6‐31+G(d,p)//AMBER (r2>0.98) and free energy corrected values (r2>0.96), finding a strong correlation (Table S2). Following suggestions from the reviewers, additional single point calculations were obtained at the M062x/6‐311+G(d,p) level and were found to be in good agreement with the former single point energies (r2>0.99, Figure S8). We henceforth refer only to the vibrational corrected single point energies unless otherwise stated. Imaginary frequencies obtained for transition state are given in Table S3.

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Figure 4 Illustration of the QM/MM optimized stationary points obtained for the ketoenamine tautomer. Key distances for each structure are reported in Angstroms (Å).

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Figure 5 Illustration of the QM/MM optimized stationary points obtained for the enolimine tautomer. Key distances for each structure are reported in Angstroms (Å).

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Table 1 Key distances associated with stationary points of the SHMT catalyzed reaction for the enolamine process. Distances are reported in Å.

Structure REACT TS1 INTA TS2 INTB TS3‐I (II) INTC‐I (II) TS4‐I (II) INTD TS5 PROD

d1 1.55 2.99 2.96 2.99 4.08 4.06 (4.10) 4.04 (4.08) 4.49 (4.63) 5.11 5.46 6.36

d2 2.26 1.32 1.02 1.32 1.70 1.70 (1.70) 1.69 (1.70) 1.69 (1.77) 1.84 1.81 1.81

d3 3.28 3.05 2.62 3.04 1.53 1.50 (1.49) 1.46 (1.46) 1.40 (1.30) 1.28 1.33 1.45

d4 5.37 5.05 4.42 5.05 3.28 3.07 (3.16) 3.14 (3.20) 2.88 (3.02) 2.84 2.04 1.52

d5 1.40 1.21 1.22 1.21 1.37 1.41 (1.40) 1.43 (1.42) 1.49 (1.97) 2.89 2.88 3.08

d6 1.77 1.79 1.80 1.79 1.74 1.69 (1.72) 1.69 (1.71) 1.73 (1.70) 1.70 1.72 1.78

d7 0.99 1.90 1.78 1.91 0.98 0.98 (0.97) 0.98 (0.97) 1.04 (0.98) 0.98 0.98 0.97

d8 2.44 2.42 2.47 2.42 2.42 3.02 (1.23) 3.51 (0.98) 3.26 (1.79) 3.83 3.89 4.74

d9 2.49 2.44 2.43 2.44 2.19 1.32 (3.06) 1.04 (3.93) 1.40 (3.64) 5.68 5.09 3.43

1Distances: d1 = PLPA‐C2‐‐‐PLPA‐CH OH, d2 = H O ‐‐‐His129, d3 = THF—N5‐‐‐PLPA‐CH OH, d4 = THF—N10‐‐‐PLPA‐CH OH, d5 = H O ‐‐‐H O, 2 2 2 2 2 2

d6 = PLPA‐N‐‐‐Asp208, d7 = H2O ‐‐‐H2O, d8 = THF‐N5H‐‐‐ THF‐O4’, d9 = THF‐N5H‐‐‐THF‐N10.

3.1 QM/MM SHMT Michaelis Complex Asp208 was simulated in the deprotonated form based on Propka predictions. However, QM/MM optimization in both the ketoamine or enolimine form led to the immediate transfer of a proton from the pyridyl nitrogen to Asp208. This is likely due to a combination of the large QM region employed coupled with the electrical embedding of the atomic charges of protein residues in the MM region. We also observed that a proton from Lys237 preferentially transferred to carboxyl group of PLP aldimine following optimization. This is consistent with the role of Lys237 which acts as the nucleophile that forms the external aldimine product in an earlier step in the SHMT. 77‐78 Lys237, His211 and Arg371 form strong H‐ bonds to the PLP substrate, as well as His129, which is involved in ‐stacking. The calculations reveal that 14


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the direct inclusion of the charged active site residues into the QM region dramatically affect the pKa of the active site residues in SHMT. In earlier QM studies on SHMT, pyridine was assumed protonated and the catalytic Asp208 residue was excluded. Furthermore, only Arg371 was included in the model. It appears that protonation of the PLP substrate in the SHMT Michaelis complex is undesirable due to repulsive interactions with 3 positively charged residues directly adjacent to it as well as less effective ‐ stacking interaction with His129. Our QM/MM calculation using a large active site representation suggests that the ketoenamine tautomer is 3.8 kcal/mol higher in energy than the enolimine form. Theoretical calculations of other PLP analogs in the gas‐phase generally point to keto form of PLP being preferred. However, studies in aqueous solution or in enzymatic systems point to the energies of both tautomers being much closer.42, 79 Our findings are consistent with experimental observation on the PLP‐dependent protein aspartate aminotransferase where neutron diffraction studies showed that when both pyridyl and imine nitrogens were deprotonated, the Schiff base pyridyl ring torsion angle was found to be coplanar.38 Indeed, we observed a planar dihedral angle in our optimized QM/MM model, which is consistent with the SHMT structure of PLP‐(L)serine aldimine.33 It should be noted that our results differ from those of Soniya et al40 on SHMT. Their research suggested that the enolimine form was 2.7 and 6.7 kcal/mol less favorable than the ketoamine form for the internal and external aldimine substrates, respectively. Crucially, the latter authors employed relatively small QM models (68 vs 198 atoms here) and simulated PLP with pyridine protonated throughout.

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Arg371

Arg371

INTA

REACT HN

O

O

N

R

H

O N

NH

O N

NH

HN H

H

H

O

HO

N

O

2-

R

(OPO3)

H

N

N

N

O

C

H

H

2-

(OPO3)

O

H H O H H N+

H

NH His129

H

H

N

H

O HO

N

O

NH

HO

NH

HN

O

Asp208

O H

O–

NH2

TS1

O

H

N

++ NH2

HO

+

NH2

Asp208

H

O

NH2

+

HN

NH+2

+ NH2

N

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NH

His129

TS2 Arg371

Arg371

INTC-I

INTB

+

HN

NH2 ++ O H NH2 NH2 HO O– N N NH O R

HN

O N

NH +

O

N

O HO

N

TS3-I

H

O

H

R

2-

(OPO3)

O N+ CH2

HN

O

H

H

H H

NH2 ++ O H NH 2 NH2 O– HO N N NH

Asp208

O

CH2

+

HN

NH

H

O

H

N N

HO

H 2-

H

H

O

(OPO3) H N

His129

NH

O N

O

H

H

Asp208

NH

His129

TS4-I Arg371

Arg371

INTD

PROD +

HN

HN

NH2 ++ O H NH2 NH2 HO O– N H N NH O R

O

HN N+

NH

H

NH2 N

O HO

N

O

TS5

2-

H H

O

O

+

NH2 ++ O H NH2 HO O– N H

(OPO3)

N+ R

H N NH

His129

2-

H H

O

(OPO3) H N

O

O HO

H O

CH2 H

HN

Asp208

N

H O

N

O

CH2

NH

HN

H

N H

Asp208

NH

His129

Scheme 3 2D representation of the lowest energy pathway associated with the SHMT catalytic process. The key residues involved in the process and truncated ligands are shown for the purpose of clarity.

3.2 SHMT Catalytic Mechanism An investigation of the retro‐aldol and direct displacement reactions for both the enolimine and ketoamine Michaelis complex structures were undertaken. Experimental observations have confirmed that the rate determining step in the SHMT catalytic reaction is the formation of formaldehyde in the first step.31‐32 Efforts to obtain a pathway involving a direct displacement were not successful due to the sizeable distance between the N5 of THF and the Cof the PLP‐serine aldimine substrate (~3.3 Å). 16


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Our investigation led to the identification of a mechanism consistent with a modified retro‐aldol process involving five steps (Scheme 3). The first step sees the breaking of the C‐C bond which necessitates the loss of a proton to a proximal basic residue. We observed that the enolimine based process resulted in lower barrier to reaction than the ketoenamine for this, the rate determining step (Figure 6). Henceforth, we shall focus our discussion on the former tautomer (Table S2). The second step identified involved the nucleophilic attack of the formaldehyde C atom by the N5 of THF. This was followed by the intramolecular proton transfer which could occur in two different ways. Next, the formation of an iminium cation intermediate was predicted (step 4) followed by cyclization of N10 with the methylene group at N5 atom (step 5) (Figure 7). In step 1, a quinoid aldimine was formed by the transfer of a proton from the serine hydroxyl group to His129. Proton transfer was found to be facilitated by shuttling via 2 active site water molecules similar to phosphobase methylation in PfPMT57 and β‐hydroxy amino acid degradation in AxDTA, both of which are PLP‐dependent enzymes.80 The serine C‐C bond distance increases from 1.54 Å in the reactant to 2.99 Å in TS1 indicating the C‐C bond is broken well before proton transfer has occurred (Table 1). The imaginary frequency associated with TS1 is understandably therefore dominated by proton transfer to histidine. The proton is located 1.32 Å from the histidine H. The sum of the Mulliken charges on His129 during step 1 increases considerably from ‐0.02 in reactant to 0.61 in TS1 (Table S1). The corresponding Mulliken charge of the PLP aldimine quinoid intermediate decreases from ‐0.07 in the reactant to ‐0.86. This also leads to a small increase in charge transfer to residues His211, Lys237 and Arg371. The N5‐‐‐ C(CH2O) distance is found to be 2.62 Å and the C‐‐‐C(CH2O) being 2.96 Å. The barrier to reaction for step 1 was predicted to be 16.6 kcal/mol for the enolimine tautomer and 18.7 kcal/mol for the ketoamine (Figure 6). The barrier associated with C‐C bond breaking in SHMT are in good agreement with experimental Ea values obtained for related substrates at htSHMT (17.04 ± 0.6 – 19 ± 0.4 kcal/mol)32 and Ea and for mjSHMT (16.7 ± 0.5 kcal/mol).81 The results are also broadly consistent with reports of similar reactions in the literature. QM/MM metadynamics simulations of the retro‐aldol reaction catalyzed by pyruvate class II aldolase predict a barrier to C‐C bond cleavage of ~14 kcal/mol.82 QM/MM studies in 2,4′‐Dihydroxyacetophenone dioxygenase enzyme estimate C−C bond breaking to be 24.9 kcal/mol83 and in (2R, 3S)‐dimethylmalate lyase (DMML) C‐C bond breaking requires 17.3 kcal/mol.84 QM/MM studies of the dehydration of methanediol giving formaldehyde displayed a barrier of approximately 20 kcal/mol.85

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Although the ketoamine form of the PLP substrate is also able to undergo reaction to form the quinoid intermediate, however it is less favourable than the enolimine form. Firstly, the Michaelis complex of the enolimine tautomer is 3.8 kcal/mol lower in energy and its rate determining barrier is lower by 2.1 kcal/mol. This would appear to suggest that the former will play a more important role in the SHMT catalyzed reaction. We have therefore neglected to study the ketoenamine tautomer beyond the first two steps (which includes rate determining step) due to its lower relevance. Furthermore, it can be assumed that it reacts in an analogous manner to the ketoamine tautomer given that the subsequent steps involve the THF substrate alone.

Figure 6 Reaction profile associated with the rate determining step for the enolimine and ketoenamine tautomers with His129 acting as a general base.

X‐ray crystallization studies support the role of His129 (His122 in bsSHMT and His83 in threonine aldolase) as the general catalytic base in the retro‐aldol mechanism.30, 47 Nevertheless, Glu56 is another residue that has been proposed as a candidate in the catalytic reaction. Glu56 was added as an explicit residue in the QM region of our QM/MM model and rotated from its original position in the 4OYT crystal structure to face the Serine hydroxyl group in order to investigate this issue further (Figure S9). The breaking of the C‐C bond in the PLP aldimine substrate and proton transfer was subsequently simulated (Figure S10). The results showed a very low barrier to reaction inconsistent with step 1 being the rate determining step.31 Furthermore, the resulting quinoid intermediate displayed a strong interaction between the N5 of THF and protonated carboxylate. This resulted in a much poorer interaction between

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the N5 of THF and the formaldehyde molecule (3.1 Å vs 2.6 Å when compared with His129 acting as the base). While proton transfer to Glu56 is low (7.5 kcal/mol), further reaction of the substrates to give the required 5,10‐methylenetetrahydrofolate product is not possible (Figure S11). Mutational studies suggest Glu56 is not critical for SHMT function43, 48 and structural data that suggest it may already be protonated within the active site30, 35 rather than being in the required deprotonated form.27 Step 2 involves the condensation of formaldehyde at N5 of the THF substrate and results in a barrier of approximately 1 kcal/mol. The transition state involves the attack of the formaldehyde carbonyl by the N5 of THF leading to the formation of 5‐hydroxymethyl‐THF (INTB) The N‐‐‐H distance associated with proton transfer from Histidine shows the N‐H distance increases from 1.02 Å in INTA to 1.32 Å at the transition state. The corresponding N5—C distance decreases from 3.04 Å to 2.62 Å indicating a concerted process. In INTB, proton transfer is complete and the N5‐C bond is 1.53 Å. The Mulliken charge of 5‐ hydroxymethyl‐THF is found to be 0.85 A.U., compared to 0.11 in INTB, as a result of the formation of the tertiary ammonium group. INTB is found to be ‐7.67 kcal/mol lower in energy than the reactant. Next, we investigated the sequence of events leading to the formation of the iminium cation intermediate (INTC). In step 3 it was found that the required proton transfer and water liberation processes could not occur in a concerted manner. This step required proton transfer from the N5 of THF to a proximal base for which two possibilities existed. The first of these was intramolecular proton transfer from N5 atom of 5‐hydroxymethyl‐THF to two possible intermolecular basic centers. INTC‐I can form through the transfer of a proton from N5 of THF to the amine (N10) of pABA. INTC‐II can form by the transfer of a proton to the carbonyl group at the 4‐position resulting in an enol. It was found that INTC‐I was considerably more favourable than INTC‐II on account of the weaker basicity of C4 carbonyl. The predicted barriers to reaction were found to be 1.1 vs 12.6 kcal/mol, both of which are lower than TS1 (Figure 7). The sum of Mulliken charges on THF for structures involving pathway–I are generally lower as a result of the more effective delocalization of charge to adjacent active site residues, particularly His129. INTC‐I was found to be 4.8 kcal/mol lower in energy than INTB.

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Figure 7 Reaction profile associated with the rate determining step for the enolimine tautomer with His129 acting as a general base. Two possible routes leading to the interconversion of INTB to INTD are feasible.

Step 4 requires the protonation of the methylene hydroxyl group and the loss of water thereby leading to the iminium cation intermediate. Again, two possible pathways to INTD exist, one from INTC‐I and the other from INTC‐II. The process involving the C4 enol is markedly higher in energy than the N10 anilino group with barrier of 5.8 vs 4.0 kcal/mol respectively. Again, more effective delocalization of charge within the active site was observed in the latter. Finally, step 5 requires the nucleophilic attack of the 5‐iminium cation in INTD by the anilino N10 atom to form the 5,10‐methylene tetrahydrofolate product. The N‐‐‐C distance is found to be 2.84 Å at INTD and 2.04 Å at the transition state (TS5). The barrier to reaction is found to be 13.0 kcal/mol, which is 3.6 kcal/mol lower in energy compared to the rate determining TS1. The process results in a slight translation of pABA ring on N‐C bond formation (1.52 Å), altering its position somewhat within the hydrophobic cavity and its interactions with the atoms of Tyr63, Phe134 and Phe266. While Lys237 was found to be slightly more stable than deprotonated form by 2.7 kcal/mol.

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4.0 Conclusions The reaction mechanism of SHMT is to‐date not fully understood despite a wealth of structural and biochemical information. We utilized a QM/MM based model of the SHMT dimer protein with a QM region of 198 atoms representing 12 residues and the two substrates and explicit solvent. We investigated the catalytic mechanism of SHMT as well as the protonation state of pyridyl ring in the PLP‐substrate, the relative energies of the ketoamine and enolimine tautomers. The catalytic reaction in SHMT is predicted to follow a retro‐aldol synthetic process with His129 acting as the general base in the reaction. The PLP‐serine aldimine and THF substrates, each undergoes reaction in SHMT in a separate, sequential process. The first step, and rate determining step, sees the breaking of the C‐C bond in the PLP‐serine aldimine substrate leading to the formation of formaldehyde. This observation is in line with experimental observations31, 34 and the predicted barriers of ~ 16 kcal/mol are similar to experimental reports of ~17‐19 kcal/mol.32, 81 Our results suggests that His129 the general base in the catalytic reaction,30, 32, 43‐44 facilitated by active site bound water molecules which form a water network as observed in other PLP‐dependent proteins.80 We observed that Glu56 was able to catalyze the breaking of the C‐C bond, the resultant intermediate was not in a configuration capable of forming the final product 5,10‐methylene tetrahydrofolate. Nevertheless, it may help to explain the weak residual activity displayed in some His129 mutant enzymes.86 Finally, we found that Asp208 is preferentially protonated in the Michaelis complex and that the enolimine tautomer was lower in energy than the ketoamine form. These observations appear to be due to the inclusion of protein charges surrounding the active site directly in the QM calculation via electrical embedding. The findings reported herein on the catalytic mechanism of SHMT could prove useful in the ongoing efforts to develop inhibitors of SHMT. Indeed, the design of inhibitors of in inhibitors for folate enzymes such as DHPS and DHFR have relied heavily on the exploitation of structural knowledge of the reaction mechanism and their natural substrates to design tight binding substrate‐analog inhibitors. 15, 21‐23

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

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Associated content includes an illustration of the SHMT active site models, RMSD and distances observed during the MD simulations, correlation between single point energies at the M062x/6‐ 31+G(d,p) and the M062x/6‐311+G(d,p) levels, optimized geometries and QM/MM energy profiles obtained using the enolimine substrate in which Glu56 acts as the general base, tables detailing the Mulliken charges on the QM residues of stationary points and imaginary frequencies associated with the transition states.

Acknowledgments MPG would like to acknowledge financial support from the Thailand Research Fund (RSA6180073) and King Mongkut’s Institute of Technology Ladkrabang. JS would like to acknowledge financial support from the Development and Promotion for Science and Technology talents project (DPST, Thailand). All authors acknowledge the National e‐Science Infrastructure Consortium for providing computing resources that have contributed to the research results reported within this paper. URL:http://www.e‐science.in.th.

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Theoretical Evaluation of the Reaction Mechanism of Serine

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