Mutation of active site glutamate in serine hydroxymethyltransferase allows trapping a reactive intermediate: a combined neutron and X-ray crystallography study

Abstract

Serine hydroxymethyltransferase (SHMT) is a pyridoxal-5′-phosphate (PLP) dependent enzyme that catalyzes a chemical transformation essential for the one-carbon (1C) metabolism. SHMT reversibly converts L-Ser into Gly and transfers a 1C unit to tetrahydrofolate (THF) to give 5,10-methylene-THF (5,10-MTHF). 5,10-MTHF, a 1C-unit donor, plays a crucial role in the downstream biomolecular syntheses required for the cell homeostasis and proliferation. SHMT is a prominent target for the drug discovery to battle bacterial and parasitic infections, and to treat various types of cancer. SHMT-catalyzed chemistry is governed by the general acid-base catalysis. Knowledge of the catalytic mechanism can aid drug design but can only be achieved when the atomic details of each reaction step are mapped, including accurate determination of hydrogen atom positions. Here we utilized the inactive E53Q mutant of Thermus thermophilus (Tth) SHMT to directly determine protonation states with room-temperature neutron crystallography and to capture a reactive intermediate containing the PLP-L-Ser external aldimine and THF in the enzyme active site. We observed protonation of the Schiff base nitrogen (N_SB) in the PLP internal aldimine but no change in the protonation states of other ionizable PLP groups and active site residues compared to wild-type TthSHMT. X-ray structural analysis of the ternary intermediate complex E53Q-Ser-THF that eluded previous structural characterization shows the strategic positioning of the E53Q side chain in close proximity to the external aldimine and THF and reinforces the proposed role for E53 as the driver of proton transfer events along the reaction pathway.

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Introduction

Serine hydroxymethyltransferase (SHMT), an enzyme at the forefront of the one-carbon (1C) metabolic pathway, is a fold-type I pyridoxal-5′-phosphate-dependent enzyme responsible for the production of 5,10-methylene-tetrahydrofolate (5,10-MTHF), which is a reactive 1C-donating biomolecule. SHMT accomplishes the synthesis of 5,10-MTHF by converting L-serine into glycine and a concurrent transfer of the 1C unit (i.e. Cβ of L-Ser) to tetrahydrofolate (THF) in the THF-dependent reaction^1–3 (Fig. 1). Recently, it has been shown that SHMT also possesses THF-dependent dehydratase activity on D-serine, that is degraded into pyruvate and ammonia.^4,5 In addition, SHMT can catalyze the retro-aldol cleavage of some β-hydroxy amino acids, decarboxylation of aminomalonate, and racemization and transamination of L- and D-alanine in a THF-independent manner.^6,7 Downstream of SHMT along the 1C metabolic pathway, 5,10-MTHF is utilized by thymidylate synthase to directly transfer the 1C unit to deoxyuridine monophosphate (dUMP) to make deoxythymidine monophosphate (dTMP),⁸ by a methylenetetrahydrofolate reductase to synthesize 5-methyl-THF,⁹ and by a methylenetetrahydrofolate dehydrogenase to create 5-formyl-THF.¹⁰ These metabolic products (Fig. 1) are critical for the subsequent biosyntheses of DNA, methionine, S-adenosylmethionine, formyl-methionyl-tRNA, and other essential biomolecules.^11–13 Recent advances in our understanding of SHMT's vital role in cell homeostasis and proliferation have propelled it to become a prominent target for the discovery of novel small-molecule antibacterial, antiparasitic, and anticancer drugs.^13–19

Fig. 1 Chemical diagrams of THF and its metabolic derivatives, PLP in its internal and L-Ser and Gly external aldimine forms. Only 6(S) diastereomers of THF and its derivatives bind to the SHMT active site. Red arrows indicate chemical groups whose protonation states were determined in the wild-type TthSHMT with neutron crystallography.

To advance the design of SHMT inhibitors, including mechanism-based compounds, understanding SHMT catalysis at the atomic level is of paramount importance. The SHMT catalytic mechanism has been debated for over 40 years,^1,20–23 with a number of mechanisms proposed. No consensus has been reached, however, mainly due to the inability of the biophysical methods employed, such as X-ray crystallography, to determine the positions and movement of hydrogen (H) atoms, which led to contradictory results from computational studies.^24–26 H atoms are key players in the general acid-base catalysis that governs SHMT function. H atom positions define the protonation states and establish the electrical charges on specific chemical groups, thus controlling the active site electrostatic environment and thereby directing enzyme catalysis. Neutron crystallography^27,28 is a powerful technique capable of pinpointing positions of functional H atoms involved in enzyme-driven proton transfers and labile H atoms that can move upon changes in the chemical and electrostatic environment inside the active site, and also accurately orienting and visualizing water species.^29–32 The reason neutron crystallography can directly determine locations of H atoms in a biological macromolecule at resolutions as low as 2.5–2.6 Å^33–35 is due to the fundamental scattering properties of neutrons. Neutrons are scattered by the atomic nuclei rather than by electron clouds that scatter X-rays, resulting in the atomic neutron scattering power being independent of the atomic number (https://www.ncnr.nist.gov/resources/n-lengths). H and its heavier isotope deuterium (D) scatter neutrons as well as C, N, and O. In addition, the cold neutrons with wavelengths of 1–5 Å, utilized in macromolecular crystallography, are nonionizing and do not induce radiation damage in crystals. This allows neutron diffraction data collection to be carried out at near-physiological (room) temperature.

We have been employing room-temperature neutron crystallography to investigate the catalytic mechanism in a bacterial SHMT from Thermus thermophilus (TthSHMT, Fig. 2A). TthSHMT is used as a model enzyme for the human mitochondrial SHMT2 (hSHMT2), because the active sites of the two proteins are conserved.^36–38 We have previously obtained five neutron structures of TthSHMT, capturing the enzyme at various stages of its catalytic process, including (1) the apo-form that lacks the pyridoxal-5′-phosphate (PLP) coenzyme entirely, (2) the holoenzyme with PLP bound to the catalytic K226 in the internal aldimine form, (3) a pre-Michaelis complex with L-serine (L-Ser) coordinated at the peripheral binding site, (4) a complex with folinic acid mimicking the carbinolamine-THF intermediate, and (5) a ternary complex containing PLP-glycine (PLP-Gly) external aldimine and 5-methyl-THF (Fig. 1). Nuclear density maps revealed the positions of all exchangeable H atoms, provided accurate assignments of the protonation states and permitted us to propose a catalytic mechanism for both stages of the reaction – the initial conversion of the PLP internal aldimine into the external aldimine upon L-Ser attack on the Schiff base and the transfer of L-Ser Cβ-OH to THF with its subsequent dehydration leading to the product 5,10-MTHF (Fig. S1). In short, during the first stage, L-Ser substrate enters the active site as a zwitterion, with its amino group protonated and carboxyl group deprotonated. L-Ser initially binds at the peripheral binding site and the gating loop consisting of residues 342–356 in TthSHMT is in the open conformation (Fig. 2B). L-Ser then moves deeper into the active site interior and binds at the cationic binding site near PLP. To become a reactive amine, the L-Ser N-terminus protonates the neutral Schiff base and subsequently attacks the Schiff base C4′, resulting in the PLP-L-Ser external aldimine formation through the transaldimination reaction. In the next stage, THF binds to the peripheral binding site and its position is secured by the gating loop closure. The L-Ser Cβ-OH group is transferred to THF via a direct attack by the THF's N5 activated through its deprotonation by the catalytic glutamate (E53 in TthSHMT). The result is the formation of the THF carbinolamine and the PLP quinonoid intermediates. The carbinolamine is dehydrated when a proton is transferred from the protonated E53 to its OH group, converting this intermediate into the THF N5-iminium cation. The iminium intermediate is cyclized to give the product 5,10-MTHF through the N10 attack on CH₂ after N10 is deprotonated by E53. Concurrently, the PLP quinonoid intermediate is converted into the PLP-Gly external aldimine by K226 protonating the PLP-Gly Cα. The PLP internal aldimine is regenerated through a transaldimination reaction utilizing the K226 side-chain amine, similar to the reaction involving L-Ser, to give the product Gly. Thus, in our proposal E53 acts as a universal acid-base catalyst in the direct displacement mechanism for the THF-dependent reaction catalyzed by SHMT,³⁷ unlike in previously proposed catalytic mechanisms that suggested involvement of several different residues.^20,23,24,39

Fig. 2 Three-dimensional structure of TthSHMT wild-type in complex with THF. (A) Homodimeric TthSHMT in cartoon representation. PLP internal aldimine and bound THF are shown as sticks in protomer B (PDB ID 9O50). (B) Superposition of TthSHMT-THF complex and the enzyme's apo-form (PDB ID 9O5G) highlighting the locations of the substrate binding sites and the gating loop motion upon THF binding.

A crucial role of a catalytic glutamate in the THF-dependent catalysis of SHMT has been reported previously. It was shown that mutation E74Q in sheep liver cytosolic SHMT (scSHMT) and E75Q in rabbit liver cytosolic SHMT (rcSHMT) enzymes reduced the 5,10-MTHF production by 400–500 times, whereas E75L substitution in rcSHMT completely abolished the THF-dependent catalysis.^39,40 Interestingly, in a bacterial SHMT from Bacillus stearothermophilus (bsSHMT) mutation of the conserved glutamate to glutamine (E53Q) inactivated the enzyme completely,⁴¹ in agreement with its proposed role as the general acid-base catalyst.³⁷ The location of the catalytic glutamate is identical in the TthSHMT sequence to that in bsSHMT. We therefore aimed to map the H atom positions in the TthSHMT E53Q mutant, compare them with the wild-type holoenzyme, and trap a reactive intermediate containing PLP-L-Ser external aldimine and THF. Here, we present a 2.4 Å resolution room-temperature neutron structure of the TthSHMT E53Q holoenzyme containing the PLP-K226 linkage in its internal aldimine form. Moreover, we succeeded in trapping the E53Q mutant in a reactive complex with the PLP-L-Ser external aldimine and THF bound at the peripheral binding site, referred to as E53Q-Ser-THF henceforth, and obtained its room-temperature X-ray structure at 1.8 Å resolution. In the neutron structure of E53Q holoenzyme, we observed, for the first time in TthSHMT, the protonated internal aldimine Schiff base, whereas the rest of the protonation states within the active site remained unchanged compared to the wild-type holoenzyme. In E53Q-Ser-THF, Q53, L-Ser Cβ-OH and THF's N5 and N10 are correctly juxtaposed for catalysis, but the lack of the general base necessary to deprotonate N5 due to E53Q mutation does not allow the reaction to proceed. The current neutron structure of TthSHMT depicting protonation states in the E53Q holoenzyme and the X-ray structure of E53Q-Ser-THF complex reaffirm the critical role of the catalytic glutamate in the THF-dependent SHMT catalysis and show the active site preorganization required for the catalytic reaction.

Results

Protonation states in TthSHMT E53Q and comparison with the wild-type holoenzyme

To map the protonation states in the active site of TthSHMT E53Q we obtained the room-temperature neutron structure of this inactive mutant holoenzyme at 2.4 Å resolution, jointly refined with the 1.65 Å room-temperature X-ray diffraction data collected from the same crystal. TthSHMT E53Q crystals are monoclinic, having the space group of P2₁, with the unit cell identical to that of the wild-type enzyme.³⁶ The whole homodimer of the E53Q holoenzyme occupies the asymmetric unit and the active sites in protomers A and B are the same, including the protonation states. The neutron structure analysis given below, therefore, is based on the active site in protomer A, unless specified otherwise.

In the E53Q holoenzyme, PLP is covalently connected to Lys226 Nζ atom through the Schiff base linkage. The N1 pyridine nitrogen of PLP is protonated and positively charged (Fig. 3A). N1 is hydrogen bonded to the carboxylate side chain of Asp197 with the D⋯O and N⋯O distances of 1.8 and 2.6 Å, respectively. The Asp197 side chain position is secured by additional hydrogen bonds it makes with the side chains of Asn98 and His125, and with the Ala199 main chain. The O3′ oxygen atom is in the deprotonated phenolate form. O3′ makes an unconventional C–H⋯O bond with Cδ2 of His200 imidazole (H⋯O and C⋯O distances of 2.1 and 3.0 Å, respectively). Such His200 orientation allows it to form a hydrogen bond with His312 having the D⋯N and N⋯N distances of 2.1 and 3.1 Å, respectively, and a bifurcated N–D⋯O hydrogen bond with SO₄²⁻ anion, with the D⋯O distances of 2.2 and 2.4 Å and N⋯O distances of 3.0 and 3.3 Å. His312 side chain rotation is controlled by the hydrogen bonds with its own main chain amide ND and with His200.

Fig. 3 Neutron scattering length density maps for the active site in TthSHMT E53Q holoenzyme. The 2F_O−F_C map shown as light brown mesh is contoured at 1.2 σ level and omit F_O−F_C map in red mesh is contoured at 2.5 σ level. (A) PLP internal aldimine and surrounding residues. The out-of-plane rotation of the Schiff base is 14° depicted using orange sticks (the equivalent torsion angle in protomer B is 17°). (B) Binding of a sulfate anion and the mutation site. (C) Superposition of the wild-type (PDB ID 8SUJ) and E53Q mutant holoenzyme active sites. H atoms are colored white, and D atoms are green. Orange arrows indicate shifts and rotations of specific groups due to mutation. Distances are in Angstroms.

The Schiff base nitrogen N_SB of PLP was found to be protonated, hence, positively charged. The omit F_O−F_C neutron scattering length density map unambiguously demonstrates the presence of D on N_SB (Fig. 3A). Thus, N_SB gained a D atom in the E53Q mutant but was observed in the neutral (non-protonated) form in the PLP internal aldimine state of the wild-type holoenzyme and in complexes with L-Ser or folinic acid bound at the peripheral binding site.^36,37 The protonated N_SB was also detected in the PLP-Gly external aldimine structure of the wild-type enzyme, presumably protonated by the incoming zwitterionic Gly substrate.³⁸ The N_SB–D bond faces O3′ making a 1.7 Å hydrogen bond and creating a six-membered cycle that stabilizes the Schiff base position. The protonated C4′=N_SB double bond is 14° (protomer A) and 17° (protomer B) out of the pyridinium plane on the si face of PLP. The corresponding dihedral angles were measured at 29–38° for the neutral Schiff base of the PLP internal aldimine in our earlier neutron structures.^36,37 This observation is in agreement with our previous quantum-chemical calculations that showed the out-of-plane geometry of the neutral Schiff base is stabilized by hyperconjugation with the pyridinium ring, whereas the N_SB protonation promotes the C4′=N_SB double bond rotation towards the pyridinium plane.^36,42

In the current neutron structure of E53Q holoenzyme, a sulfate anion (SO₄²⁻) is observed captured in the cationic binding site. The SO₄²⁻ anion position is stabilized by a favorable electrostatic interaction with the positively charged R358 guanidinium side chain (Fig. 3B and S2). The sulfate is further surrounded by H122, H200, Q53*, and Tyr61* (the asterisk indicates the residue belongs to protomer B) and makes a water-mediated interaction with Q53*. Importantly, the protonation states of all active site histidine residues, including H122, H125, H200, H225, and H312 (Fig. S3), remain neutral and identical tautomers with Nε2 nitrogen atoms protonated and Nδ1 not protonated, as was also observed in our previous neutron structures of TthSHMT.^36–38

The TthSHMT wild-type and E53Q mutant holoenzyme neutron structures superimpose with the RMSD of 0.12 Å, implying the catalytic residue mutation did not perturb the overall enzyme structure, although small structural differences within the active site are possible. The positions of most active site residues are virtually identical, including the PLP internal aldimine except for the tilt of the Schiff base (Fig. 3C). One notable difference is at the mutation site where the Q53* side chain shifts and rotates away from where the E53* side chain was placed in the wild-type enzyme to lose a water-mediated contact with H122. The other difference is a slight 0.6 Å, but significant, shift and a 30° rotation of SO₄²⁻ anion away from R358, weakening the salt bridge hydrogen bonds. The sulfate movement also results in the breaking of hydrogen bonds with Y61* and S31 side chain hydroxyls that rotate by 45° and 51° in E53Q holoenzyme.

Active site pre-organization in the reactive E53Q-Ser-THF complex

To visualize the intermediate formed just before the L-Ser Cβ-OH hydroxymethylene group is transferred to the THF's N5 atom, we succeeded in determining a room-temperature X-ray structure of the TthSHMT E53Q mutant ternary complex with L-Ser and THF at 1.8 Å resolution (Table S2). Interestingly, earlier attempts to obtain such a complex by using the inactive rcSHMT E75L mutant failed, with the electron density map found to be inadequate to determine what species were bound in the active site.³⁹ In the E53Q-Ser-THF complex, however, the electron density unequivocally showed the PLP-L-Ser external aldimine, the K226 side chain released from the starting internal aldimine, Q53, and THF at the peripheral binding side (Fig. 4A). As in our previous complexes of TthSHMT,^37,38 the PLP-L-Ser external aldimine formation and THF binding occurred only in protomer B (Fig. S4), because the gating loop open-close dynamics required for the active site reactivity towards L-Ser and THF binding are hindered in protomer A due to crystal packing interactions. The electron density also reveals that only the C6-(S) diastereomer of THF is bound in E53Q-Ser-THF, as it does in the TthSHMT/THF complex.³⁸

In E53Q-Ser-THF, the PLP-L-Ser external aldimine is held in place by numerous conventional and nonconventional interactions (Fig. 4B). The pyridine nitrogen makes a short 2.5 Å hydrogen bond with D197, whereas the O3′ forms a nonconventional C–H⋯O hydrogen bond with H200. The C-terminal carboxylate of L-Ser takes advantage of all hydrogen bonding possibilities by forming a salt bridge with R358 and hydrogen bonds with Y61* and H200. Several hydrogen bonds made by the phosphate group with the main chain amides of G94*, S95* and G258* secure its position. In addition, the pyridine ring of PLP-L-Ser interacts by the π⋯π stacking and C–H⋯π contacts with the side chains of H122 and A199, respectively. The Schiff base in the external aldimine is rotated by −10.5° out of the pyridine plane, which is similar to the C4′ = N_SB out-of-plane torsion of −8° in TthSHMT-Gly-5MTHF complex.³⁸ In both complexes the Schiff base rotates past the pyridine plane to the re face in the PLP-Gly external aldimine (Fig. S5). Such a PLP-L-Ser external aldimine conformation suggests protonation of N_SB as observed in the PLP-Gly external aldimine and ensures that the Cβ-OH group is close to THF's N5 making a 2.6 Å hydrogen bond. The K226 side chain amine is placed above the Schiff base as in the PLP-Gly external aldimine and its Nζ atom makes a 3.6 Å N–H⋯π contact with C4′ (Fig. 4B). Importantly for the catalytic mechanism, N5 is just 3.6 Å away from PLP-L-Ser Cβ and 3.8 Å away from Q53* side chain. Q53* also contacts Cβ, with the O⋯C distance of 3.2 Å, and is just 3.7 Å away from the THF's N10.

Discussion

H atoms are key players in general acid-base catalysis, as the catalytic reactions are facilitated through a series of proton transfers occurring in the enzyme active sites to convert substrates into products. In THF-dependent SHMT catalysis, which involves two substrates and a covalently bound coenzyme PLP, no fewer than ten proton transfer events must happen to convert the L-Ser and THF substrates into Gly and 5,10-MTHF products, respectively. Several neutron crystallographic studies^36–38 have revealed the protonation states of the PLP in both internal and external aldimine forms, active site residues, and THF analogues. These studies established E53 as the only residue capable of changing its protonation state along the reaction pathway – an observation only possible by neutron crystallography. Crucially for catalysis, E53 is strategically placed in close proximity to the reactive Cβ-OH group of the PLP-L-Ser and THF's N5 and N10 atoms. This led us to propose a direct displacement mechanism for the THF-dependent reaction catalyzed by the conserved glutamate (E53 in TthSHMT and bsSHMT) acting as a universal acid-base catalyst, instead of by several different residues.³⁷ We therefore engineered the point mutation E53Q in TthSHMT in order to study the inactive mutant's active site organization at the atomic level and to trap a reactive intermediate that eluded structural characterization previously.

Unsurprisingly, in the E53Q holoenzyme, the pyridine N1 atom of the PLP internal aldimine is protonated, thus positively charged, as it is hydrogen bonded to the Asp197 carboxylate, a conserved residue in fold type I PLP-dependent enzymes (Fig. S6).^36,42–44 O3′ is observed as a deprotonated, negatively charged phenolate that engages in an unconventional C–H⋯O bond with the H200 imidazole. The O3′ protonation state is unchanged in previous wild-type TthSHMT neutron structures,^36–38 and in aspartate aminotransferase (fold type I)^42,45 and tryptophan synthase (fold type II).⁴⁶ Five active-site histidines, His122, His125, His200, His225, and His312, create a histidine cage that encapsulates the PLP. All of these histidines are neutral in E53Q, with protonated Nε2 and non-protonated Nδ1, and their protonation states and tautomeric forms are invariant between various complexes and reaction intermediate mimics of wild-type TthSHMT.^36–38 The neutron scattering length density map also revealed that the Schiff base N_SB is protonated in E53Q holoenzyme. This is the first time the protonated N_SB is observed for the PLP internal aldimine in TthSHMT, suggesting the Schiff base protonation state can be finely tuned by the active site electrostatic environment. Computational studies will be required to support the proposed mechanism for SHMT catalysis³⁷ that involves protonation of the Schiff base by an incoming amino acid substrate.

Addition of L-Ser and THF to E53Q mutant enzyme resulted in the formation of the PLP-L-Ser external aldimine and THF binding at the peripheral binding site to give a ternary E53Q-Ser-THF complex. E53Q-Ser-THF is a reactive complex with the PLP-L-Ser Cβ-OH group poised for transfer to THF's N5 when the nitrogen is deprotonated. This complex eluded structural analysis as previous attempts failed to generate interpretable electron density maps using mutant bsSHMT.³⁹ In the current E53Q-Ser-THF room temperature X-ray structure, the electron density is unambiguous for the PLP-L-Ser external aldimine and THF, showing preorganization of the active site for catalysis. The Q53 side chain is naturally not capable of carrying out the necessary proton transfers for catalysis to ensue, but its position in relation to the external aldimine and THF would indicate that, in the actual catalytic E53-Ser-THF complex, the glutamate would be able to execute catalysis.

Conclusion

By using neutron protein crystallography at near-physiological (room) temperature we visualized the protonation states in the inactive E53Q mutant of TthSHMT holoenzyme. The PLP internal aldimine is protonated at both pyridine N1 and Schiff base N_SB atoms, having the C4′ = N_SB bond rotated closer to the pyridine plane than in the wild-type holoenzyme. As in all neutron structures of TthSHMT, the active site His residues are neutral, suggesting their protonation states conservation is important for creating a specific electrostatic environment for catalysis. We also succeeded in determining a room-temperature X-ray structure of the elusive substrate–bound complex containing PLP-L-Ser external aldimine and THF. Structural analysis of this complex reinforces the proposed role for E53 as the general acid-base catalyst that drives the proton transfer events along the reaction pathway. TthSHMT E53Q is a useful model system for further neutron diffraction studies of the catalytic mechanism, including for hSHMT2, and can be utilized in the neutron structure-assisted design of SHMT inhibitors.

Methods

General information

Columns for protein purification were purchased from Cytiva (Piscataway, New Jersey, USA). His-tagged Tobacco Etch Virus (TEV) protease was produced in-house. Crystallization reagents and supplies were purchased from Hampton Research (Aliso Viejo, California, USA). Crystallographic supplies for crystal mounting and X-ray and neutron diffraction data collection at room temperature were purchased from MiTeGen (Ithaca, New York, USA) and Vitrocom (Mountain Lakes, New Jersey, USA). L-Ser (cat. no. S4500) and THF (a mixture of diastereomers, cat. no. T3125) were purchased from Millipore Sigma (St. Louis, Montana, USA).

Expression and purification. A detailed procedure for the expression and purification of TthSHMT was published elsewhere.^36,37 The kanamycin-resistant pJ411 plasmid (ATUM, Newark, CA) encoding TthSHMT E53Q variant and an N-terminal linker containing a His₆-tag, a 34 amino acid long linker sequence and a TEV protease cleavage site ENLYFQS was transformed into the E.coli BL21(DE3) expression vector. The cells were grown in Luria–Bertani (LB) media with 50 µg mL⁻¹ kanamycin at 37 °C to an optical density (OD) of 0.8–1.0. For protein expression, the cell cultures were induced with 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and maintained overnight at 22 °C in a shaker. After centrifugation the next day, the cell pellets were resuspended in the lysis buffer made with 50 mM sodium phosphate pH 7.5, 500 mM NaCl, and 10 mM imidazole at a ratio of 5 mL of the buffer per gram of wet cell paste. Lysozyme was added to the resuspended cells at a concentration of 0.1 mg mL⁻¹ and stirred on ice for 30 min before the cells were disrupted by sonication. The lysate was clarified by centrifugation at 30 000 g to remove the insoluble fraction. The His-tagged TthSHMT E53Q was purified via affinity chromatography using a HisTrapFF (5 mL) nickel column equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCl, and 10 mM imidazole. The protein was eluted from the column using a linear gradient of 20 mM HEPES pH 7.5, 100 mM NaCl, and 500 mM imidazole. TEV protease (1 mg TEV protease/100 mg of tagged protein) was added to purified TthSHMT E53Q and the mixture was dialyzed against 20 mM HEPES pH 7.5, 100 mM NaCl, and 1 mM EDTA overnight at room temperature. The resultant solution was loaded onto a HisTrapFF (5 mL) nickel column and untagged TthSHMT E53Q was collected in the flow-through. Pure TthSHMT E53Q was dialyzed against 40 mM NaOAc pH 5.4 and 1 mM PLP to ensure all enzyme molecules contained active sites with bound PLP in internal aldimine form and concentrated to ∼20 mg mL⁻¹. For long-term storage at −30 °C, glycerol was added to the concentrated protein up to the concentration of 20% (v/v) as a cryoprotectant to avoid the protein crashing out of the solution upon thawing.

Crystallization, soaking, and H/D-exchange. For crystallization experiments, frozen TthSHMT E53Q was thawed and dialyzed overnight against 40 mM NaOAc pH 5.4 and 1 mM PLP to remove the cryoprotectant. The protein was then crystallized by sitting drop vapor diffusion methodology using 40 mM NaOAc pH 5.5, 1 M (NH₄)₂SO₄, and 0.5 M Li₂SO₄ as the precipitant solution at 16 °C. To obtain large crystals crystallization drops were microseeded with a seeding tool from Hampton Research in 9-well glass plates and sandwich box setups using microseeds obtained by crushing previously crystallized aggregates. A large crystal of TthSHMT E53Q was mounted in a 2 mm-inner diameter quartz capillary containing a liquid plug made of 40 mM NaOAc pH 5.5, 1 M (NH₄)₂SO₄, and 0.5 M Li₂SO₄ in 99.8% D₂O to perform H/D-vapor exchange. Because THF does not bind to TthSHMT in high concentrations of salt, the E53Q-Ser-THF complex was made in the following way. A crystal of TthSHMT E53Q holoenzyme was transferred to a fresh drop containing 40 mM NaOAc pH 5.5 and 15% PEG 4000 and soaked overnight to remove most of sulfate. The crystal was then moved into another drop containing a soaking solution with 40 mM NaOAc pH 5.5, 15% PEG 4000, 0.5 M L-Ser and 10 mM THF on the next day, and the crystal was kept in this solution overnight before harvesting it for room-temperature X-ray crystallography.

Fig. 4 Organization of the active site in protomer B of E53Q-Ser-THF complex. (A) The 2F_O−F_C electron density map is shown as violet mesh contoured at 1.4 σ level. (B) Interactions between PLP-L-Ser, THF and the active site residues. Distances are in Angstroms.

X-ray diffraction data collection and structure refinement. Room temperature X-ray diffraction data collection on TthSHMT E53Q holoenzyme and E53Q-Ser-THF complex crystals were performed on a Rigaku HighFlux HomeLab instrument equipped with a MicroMax-007 HF X-ray generator, Osmic VariMax optics, and a DECTRIS Eiger R 4 M detector at ORNL. The data were indexed and integrated using the CrysAlisPro software package (Rigaku, The Woodlands, TX), and the data were reduced and scaled in the AIMLESS program in the CCP4 software suite.^47,48 The X-ray structures were solved by molecular replacement in PHASER⁴⁹ using phases from PDB code 8SUJ and refined with phenix.refine in the PHENIX suite.^50,51 The room temperature X-ray structure of TthSHMT E53Q holoenzyme was subsequently used in joint X-ray/neutron refinement. Ligand restraints for PLP internal aldimine, PLP-L-Ser external aldimine and THF were generated with eLBOW⁵² using geometry optimized by quantum chemical calculations in Gaussian16⁵³ at B3LYP/6–31 g(d,p) level of theory. The X-ray diffraction data collection statistics are presented in Tables S1 and S2.

Neutron diffraction data collection. Neutron diffraction was first tested at room temperature on the IMAGINE^54–56 instrument located at the High Flux Isotope Reactor (Oak Ridge National Laboratory) using the broad bandpass functionality with neutron wavelengths between 2.8 and 10 Å. The neutron diffraction quality from the 0.5 mm³ crystal was considered sufficient for a full data collection using the quasi-Laue diffraction mode of IMAGINE with the neutron wavelength range of 2.8–4.5 Å. The diffraction data extended to 2.4 Å resolution. Each neutron image exposure was 24 h, and the crystal was held in a stationary position. The crystal was rotated along the vertical axis (Δφ = 8°) before collecting each successive image. The crystal orientation was changed two times by tilting the capillary with respect to the incident neutron beam to improve data completeness. In total, 32 neutron diffraction images were collected. Neutron diffraction data processing was performed with a version of LAUEGEN^57,58 modified to account for the geometry of the cylindrical image plate detector. The wavelength–normalization curve was determined using the intensities of symmetry-equivalent reflections at different wavelengths in LSCALE.⁵⁹ No explicit absorption corrections were applied. The data were scaled and merged in SCALA.⁶⁰ Neutron data collection statistics can be found in Table S1.

Joint X-ray/neutron (XN) refinement. Joint XN refinement of the TthSHMT E53Q holoenzyme was carried out using nCNS,^61,62 a patch of the Crystallography & NMR Systems (CNS)⁶³ software suite in the same manner as for our previous TthSHMT structures.^36–38 A single rigid body refinement was the first step in the refinement procedure. Several separate rounds of the atomic position, atomic displacement parameter, and D atom occupancy refinements followed. In between the rounds of the joint XN refinement, the structure was visualized in the molecular graphics program COOT⁶⁴ to confirm correct side chain modeling and direct the rotation of side chain hydroxyl, thiol, and ammonium groups as well as water molecules to construct accurate hydrogen bonding networks. Water molecules were modeled and refined as D₂O due to H/D-vapor exchange. The enzyme molecule was modeled with H atoms at non-exchangeable positions because hydrogenated protein was used in the experiment, while labile (exchangeable) positions were modeled as D atoms. After D atom occupancy refinement, the exchangeable sites were modeled based on individual site occupancies, where an occupancy of −0.56 is indicative of a pure H atom and an occupancy of 1.00 reflects a pure D atom. Sites partially occupied by both H and D atoms were given two atom records with the partial occupancies adding up to 1.00. The percentage of D atom occupancy at a specific site is calculated according to the following formula: % D = (occupancy(D) + 0.56)/1.56. Joint X-ray/neutron refinement statistics can be found in Table S1.

Author contributions

V. N. D., R. S. P. and A. K. designed the study. V. N. D. expressed, purified and crystallized the protein. V. N. D. and A. K. collected and reduced X-ray and neutron diffraction data and refined the structures. V. N. D., R. S. P., and A. K. wrote the paper.

Conflicts of interest

There are no conflicts of interest to declare.

Abbreviations

Tth	Thermus thermophilus
SHMT	Serine hydroxymethyltransferase
THF	Tetrahydrofolate
5,10-MTHF	5,10-Methylene-tetrahydrofolate
5MTHF	5-Methyl-tetrahydrofolate
hSHMT2	human mitochondrial serine hydroxymethyltransferase
PLP	Pyridoxal-5′-phosphate

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: Table S1, Fig. S1–S6. See DOI: https://doi.org/10.1039/d6ra01814a.

Crystallographic data for TthSHMT E53Q and E53Q-Ser-THF have been deposited at the PDB under accession codes 10 YN and 10 ZB, respectively and can be obtained from https://doi.org/10.2210/pdb10YN/pdb and https://doi.org/10.2210/pdb10ZB/pdb.

Acknowledgements

A portion of this research used resources at the High Flux Isotope Reactor (HFIR), a DOE Office of Science User Facility operated by the Oak Ridge National Laboratory (ORNL). The beam time was allocated to IMAGINE instrument on proposal number IPTS-31466. The Office of Biological and Environmental Research supported a portion of this research that used resources at ORNL's Center for Structural Molecular Biology (CSMB), a DOE Office of Science User Facility. ORNL is managed by UT-Battelle LLC for DOE's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. This research was supported by a grant from NIH-GMS (R01GM137008) to R.S.P. and A.K.

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