Homocysteine S-methyltransferases (HMTs, EC 2.1.1.0) catalyse the conversion of homocysteine to methionine using S-methylmethionine or S-adenosylmethionine as the methyl donor. HMTs play an important role in methionine biosynthesis and are widely distributed among micro-organisms, plants and animals. Additionally, HMTs play a role in metabolite repair of S-adenosylmethionine by removing an inactive diastereomer from the pool. The mmuM gene product from Escherichia coli is an archetypal HMT family protein and contains a predicted zinc-binding motif in the enzyme active site. In the present study, we demonstrate X-ray structures for MmuM in oxidized, apo and metallated forms, representing the first such structures for any member of the HMT family. The structures reveal a metal/substrate-binding pocket distinct from those in related enzymes. The presented structure analysis and modelling of co-substrate interactions provide valuable insight into the function of MmuM in both methionine biosynthesis and cofactor repair.

INTRODUCTION

L-Methionine (Met) is a structurally unique amino acid with a hydrophobic thioether side chain [1]. Although Met is one of the less abundant proteinogenic amino acids, it has an indispensable role in the initiation of translation [2] and commonly contributes to the hydrophobic cores of proteins [3]. Met is also the main cellular carrier of methyl groups as a key component of S-adenosyl-L-methionine (AdoMet), the universal methyl donor [4]. Met biosynthesis in bacteria, fungi and plants is well understood [5,6]. The biosynthetic pathway proceeds from aspartate via homoserine and cystathionine to L-homocysteine (Hcy), whose thiol group is then methylated to give Met [7]. Met is converted into AdoMet in a reaction mediated by S-adenosylmethionine synthetases, EC 2.5.1.6 (also known as methionine adenosyltransferases).

Plants have a unique additional reaction in which Met is S-methylated to yield S-methyl-L-methionine (SMM) by methionine S-methyltransferase (MMT, EC 2.1.1.12) [8]. SMM can serve as a methyl donor to Hcy in a reaction mediated by homocysteine S-methyltransferase (HMT, EC 2.1.1.10), an enzyme present in plants, bacteria, fungi and animals. The presence of both MMT and HMT in plants sets up a cycle (the SMM cycle) that allows SMM to serve as a storage and transport form of Met [911]. The occurrence of HMTs in bacteria, fungi and animals [7,1214] enables the use of plant SMM as a source of Met [14,15]. Additionally, almost all HMTs can also use the non-natural (R,S) form of AdoMet, which is generated in vivo by spontaneous racemization of the natural (S,S) form [16] and which cannot serve as a methyl donor for other methyltransferases [12,13]. The ability to process (R,S)-AdoMet enables HMTs to recycle it back to S-adenosyl-L-homocysteine (AdoHcy) and re-enter the methyl cycle [12,13,17]. HMT can thus serve as a repair enzyme for damaged AdoMet and this is probably its ancestral function, the ability to use SMM as methyl donor having arisen later in evolution [14]. An additional evolutionary innovation is the capacity of HMT from the selenium-resistant plant Astragalus bisulcatus to use selenocysteine as preferred methyl acceptor [18].

Escherichia coli K-12 strain MG1655 has an HMT; this enzyme is encoded by the mmuM gene, which is in a two-gene operon with the SMM transporter gene mmuP [19]. E. coli MmuM is a 310-residue protein that, like most other HMTs, can use either SMM or the ‘damaged’ (R,S)-AdoMet as methyl donor; it shows a moderate preference for SMM and cannot use (S,S)-AdoMet [14]. Surprisingly, E. coli MmuM and most other HMTs cannot use S-ribosyl-L-methionine (S-ribosylMet) as a methyl donor, although this compound is structurally intermediate between SMM and (R,S)-AdoMet (Figure 1) [14].

HMT converts L-homocysteine (Hcy) to L-methionine (Met) with unique substrate selectivity

Figure 1
HMT converts L-homocysteine (Hcy) to L-methionine (Met) with unique substrate selectivity

(A) HMT (MmuM)-catalysed methionine biosynthesis with SMM or (R,S)-AdoMet, whereas structural analogues (S,S)-AdoMet (B) and S-ribosylMet (C) are not active substrates for MmuM.

Figure 1
HMT converts L-homocysteine (Hcy) to L-methionine (Met) with unique substrate selectivity

(A) HMT (MmuM)-catalysed methionine biosynthesis with SMM or (R,S)-AdoMet, whereas structural analogues (S,S)-AdoMet (B) and S-ribosylMet (C) are not active substrates for MmuM.

MmuM and other HMTs show sequence similarity to zinc-binding enzymes: betaine homocysteine methyltransferase (BHMT, EC 2.1.1.5), which uses betaine as the methyl donor [20] and methionine synthase (MS, EC 2.1.1.13), which uses 5-methyltetrahydrofolate (5-MTHF) as the methyl donor [21]. Structural prediction indicates that HMTs contain a functional zinc-binding domain. Although several BHMT and MS structures have been reported, no structural information is available for any HMT protein. In the present study, we demonstrate the first HMT protein structure, MmuM from E. coli. Structures in the oxidized (cysteine disulfide), apo (Zn2+-free) and metallated (Zn2+-Hcy) forms provide a comprehensive view of the active-site pocket and allow insights into substrate selectivity, substrate binding and the methyl transfer mechanism for the HMT family.

MATERIALS AND METHODS

Chemicals

Unless otherwise stated, reagents and chemicals were purchased from Fisher Scientific or Sigma–Aldrich.

Cloning, expression and purification of MmuM

The gene for mmuM was amplified via PCR from E. coli K-12 MG1655 genomic DNA with the following primers: mmuM_NdeIF (5′-GCGCATATGTCGCAGAATAATCCGTTA-3′) and mmuM_XhoIR (5′-GCGCACGAGTCAGCTTCGCGCTTTTAA-3′). The PCR product was cleaved with the corresponding endonucleases and then ligated into the expression vector pET30a with a C-terminal His6-tag. The resultant plasmid was transformed into E. coli C43 (DE3) cells for expression. Cultures (1 litre) were grown at 37°C to a OD600 of 0.8 and overexpression was initiated by adding IPTG (final concentration 400 μM). Growth was continued for 25 h at 25°C, before the cells were harvested by centrifugation. Cell pellets were resuspended in 25 ml of 0.5 M NaCl and 20 mM Tris/HCl, pH 7.5, and lysed at 14 000 psi (1 psi ≈ 6.9 kPa) through a nitrogen-pressure microfluidizer cell (M-110L Pneumatic). The lysate was clarified by centrifugation at 15000 g for 20 min at 4°C. MmuM was purified by immobilized metal affinity chromatography (HisPur Ni2+ nitrilotriacetate resin, Thermo Scientific). After binding for 1 h, the resin was washed four times with 10 ml of 0.5 M NaCl, 10 mM imidazole and 20 mM Tris/HCl, pH 7.5, and the bound protein was eluted with three lots of 2 ml of 0.5 M NaCl, 250 mM imidazole and 20 mM Tris/HCl, pH 7.5. The elution fraction was further purified by gel-filtration chromatography (HiLoad 16/60 SuperDex-200 column, AKTA FPLC System, GE Healthcare) with buffer containing 150 mM NaCl, 5 mM DTT and 20 mM Tris/HCl, pH 7.5. The pooled protein was dialysed against 1 litre of 150 mM NaCl, 5 mM ZnCl2, 1 mM 2-mercaptoethanol (βME) and 20 mM Tris/HCl, pH 7.5, for 4 h, then subsequently dialysed against 1 litre of 150 mM NaCl, 1 mM βME and 20 mM Tris/HCl, pH 7.5, for an additional 4 h to remove non-specifically bound Zn2+. The protein was centrifuged to remove precipitated protein before crystallization. Protein concentration was determined by the Bradford assay using BSA as standard.

Crystallization

Initial crystal screening was performed in a vapour-diffusion sitting-drop format using commercial sparse matrix screens. Small clusters of needle crystals were identified in a condition containing 2.0 M ammonium sulfate and 0.1 M sodium acetate, pH 4.6. Optimization of salt and pH along with microseeding were performed in a hanging-drop format at 20°C. Protein (1.8 μl of a 4 mg·ml−1 solution) plus 2 μl of precipitant were mixed and balanced against 1 ml of reservoir solution. Resultant rod-shaped single crystals with a size of ∼20 μm×20 μm×100 μm were obtained in a final condition that contained 1.6 M ammonium sulfate, 10% (v/v) glycerol and 0.1 M sodium acetate, pH 4.8. Crystals of suitable size were harvested and frozen in liquid nitrogen without additional cryoprotectant. Harvested MmuM crystals were in an oxidized metal-free form. To obtain the apo form crystals, 10 mM DTT was added to the reservoir solution and equilibrated with the crystal drop for 16 h. Metallated form crystals were obtained by further soaking in 1 mM ZnCl2 and 10 mM Hcy for 5 min before harvesting and flash freezing.

Data collection and processing, and structure refinement

Diffraction data were collected on beamline 21-ID-F/G of the Life Sciences Collaborative Access Team (LS-CAT) facility at the Advanced Photon Source (APS), Argonne National Laboratory. Data were collected at 100° K with a wavelength of 0.9786 Å (1 Å=0.1 nm), integrated, merged and scaled using XDS [22] to a resolution of 1.76–2.88 Å in space group I222 or P21212, with one or two protein molecules per asymmetric unit. The phase of MmuM in space group I222 was determined by molecular replacement with MOLREP [23] using the N-terminal structure of B12-dependent MS [21] (PDB entry 1Q7M, 26% sequence identity) from Thermotoga maritima as a search model. The molecular replacement solution was refined by rigid-body refinement in REFMAC5 [24] to a Rwork of 43% and Rfree of 51%. The refined molecular replacement model, combined with an anomalous signal from zinc atoms, provided an interpretable electron density map and the atomic model was completed by several rebuilding and refinement cycles using SHELXDE [25], RESOLVE [26], ARP/wARP [27], PHENIX.REFINE [28] and COOT [29]. Zn2+ ions were added manually into the calculated anomalous difference map and occupancy and anisotropic B-factors were refined sequentially. Hcy was also added manually, adjacent to the Zn2+ ion. Our metallated crystal form has extra electron density near Cys296, which we assigned as βME. A standard distance restraint between SβME and SCys296 was added (2.05 Å) during the refinement. Water molecules were placed in the structure based on manual inspection of the 2mFo–DFc and mFo–DFc electron density maps. No TLS was used during the refinement process. The quality of the model was evaluated using a simulated annealing composite omit map. The refined co-ordinates have been deposited in the PDB (accession codes 5DML, 5DMN and 5DMM). Statistics on data collection and atomic structure refinement are given in Table 1. Structural illustrations were prepared with PyMOL (http:://www.pymol.org/).

Table 1
X-ray data collection, processing and structure refinement

Values for the outer shell are given in parentheses.

 Oxidized form with disulfide bridge Apo form Zn2+-free Metallated form with Zn2+ and Hcy 
Data collection    
 Resolution range (Å) 34.39–2.44 (2.54–2.44) 38.42–2.89 (2.99–2.89) 24.23–1.78 (1.84–1.78) 
 Diffraction source 21-ID-F 21-ID-G 21-ID-G 
 Space group I222 P2121I222 
a, b, c (Å) 78.45, 84.46, 85.82 85.82, 85.94, 79.02 78.95, 85.92, 87.66 
 Total number of reflections 58379 (6367) 82600 (7945) 141424 (13523) 
 Unique reflections 10331 (1158) 13530 (1302) 27033 (2711) 
 Multiplicity 5.7 (5.5) 6.1 (6.1) 5.2 (5.0) 
 Completeness (%) 99.76 (97.69) 99.71 (98.41) 93.40 (95.36) 
 <I/σ(I)> 15.8 (3.4) 11.9 (3.5) 14.8 (2.5) 
 Wilson B factor (Å240.7 41.6 18.1 
Rmerge 0.082 (0.49) 0.141 (0.68) 0.093 (0.60) 
Rmeas 0.090 (0.59) 0.161 (0.70) 0.104 (0.72) 
 CC1/2 0.999 (0.996) 0.996 (0.932) 0.996 (0.735) 
Structure refinement 
Rwork 0.202 (0.276) 0.225 (0.280) 0.181 (0.257) 
Rfree 0.217 (0.296) 0.270 (0.324) 0.206 (0.282) 
 Number of non-H atoms 
  Protein 2169 3932 2190 
  Ligand – 10 13 
  Water 32 13 179 
 RMS bonds (Å) 0.009 0.005 0.007 
 Root mean square angles (°) 1.26 0.92 1.12 
 Ramachandran    
  Favoured (%) 95.49 96.00 98.00 
  Outliers (%) 0.2 0.35 
 Clashscore 7.69 3.81 2.05 
 Average B factors (Å2
  Protein 47.2 42.7 23.3 
  Ligand – 39.3 37.0 
  Water 43.2 38.8 35.7 
 Oxidized form with disulfide bridge Apo form Zn2+-free Metallated form with Zn2+ and Hcy 
Data collection    
 Resolution range (Å) 34.39–2.44 (2.54–2.44) 38.42–2.89 (2.99–2.89) 24.23–1.78 (1.84–1.78) 
 Diffraction source 21-ID-F 21-ID-G 21-ID-G 
 Space group I222 P2121I222 
a, b, c (Å) 78.45, 84.46, 85.82 85.82, 85.94, 79.02 78.95, 85.92, 87.66 
 Total number of reflections 58379 (6367) 82600 (7945) 141424 (13523) 
 Unique reflections 10331 (1158) 13530 (1302) 27033 (2711) 
 Multiplicity 5.7 (5.5) 6.1 (6.1) 5.2 (5.0) 
 Completeness (%) 99.76 (97.69) 99.71 (98.41) 93.40 (95.36) 
 <I/σ(I)> 15.8 (3.4) 11.9 (3.5) 14.8 (2.5) 
 Wilson B factor (Å240.7 41.6 18.1 
Rmerge 0.082 (0.49) 0.141 (0.68) 0.093 (0.60) 
Rmeas 0.090 (0.59) 0.161 (0.70) 0.104 (0.72) 
 CC1/2 0.999 (0.996) 0.996 (0.932) 0.996 (0.735) 
Structure refinement 
Rwork 0.202 (0.276) 0.225 (0.280) 0.181 (0.257) 
Rfree 0.217 (0.296) 0.270 (0.324) 0.206 (0.282) 
 Number of non-H atoms 
  Protein 2169 3932 2190 
  Ligand – 10 13 
  Water 32 13 179 
 RMS bonds (Å) 0.009 0.005 0.007 
 Root mean square angles (°) 1.26 0.92 1.12 
 Ramachandran    
  Favoured (%) 95.49 96.00 98.00 
  Outliers (%) 0.2 0.35 
 Clashscore 7.69 3.81 2.05 
 Average B factors (Å2
  Protein 47.2 42.7 23.3 
  Ligand – 39.3 37.0 
  Water 43.2 38.8 35.7 

Models of bound methyl donors and analysis

Fitting of methyl donors into the active site was conducted with AutoDock 4.2 [31]. The methyl donors SMM, (R,S)-AdoMet or their mimics (S,S)-AdoMet and S-ribosylMet were constructed and energy minimized with Spartan '08 [32]. The position of bound Hcy molecule in the metallated form MmuM was held fixed as the methyl donors were fitted. Standard algorithms and docking procedures were used for a rigid protein and a flexible ligand in a grid covering the entire protein. The best docking poses were analysed with AutoDockTools 1.5.6 and the hydrogen bonding /π–π interactions and corresponding estimated free energy of ligand binding (ΔG) were calculated (Supplementary Table S1).

MmuM active-site mutagenesis

Mutations of residues in the MmuM active site were carried out using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer's instructions. Oligonucleotides utilized were Y71F (5′-CACTGCCAGCtttCAGGCGACGC-3′), Y71A (5′-CACTGCCAGCgctCAGGCGACGCCGG-3′), Y71T (5′-CACTGCCAGCactCAGGCGACGCCG-3′) and Y71FT169Y (plus 5′-GGCCTGCGAAtacCTGCCGAATTTTTCCGAGATTG-3′). The validity of mutagenesis was confirmed by DNA sequencing.

ITC-based activity assay

MmuM wild-type (WT) and mutant proteins were purified as described above. WT and mutant proteins, prepared concurrently, were each concentrated to 10.0 mg·ml−1 for isothermal titration calorimetry (ITC)-based kinetics assays. Protein samples (1.0 μl, final concentration 1.0 μM), along with 2 mM DL-Hcy, were mixed with 300 μl of freshly prepared reaction buffer (100 mM NaCl, 100 mM KCl, 20 mM Tris/HCl, pH 7.5, and 1 mM DTT) and injected into the MicroCal iTC200 (Malvern) reaction cell (cell volume 200 μl). 39.6 μl of 5.0 mM DL-SMM was dissolved in the same reaction buffer and titrated into the cell in the single injection mode over 800 s at 30°C (Supplementary Figure S1A). Potassium-free protein (WT) kinetics was carried out in a K+-deficient environment (200 mM NaCl, 20 mM Tris/HCl, pH 7.5, and 1 mM DTT). Protein-free blank runs were performed in a similar fashion. Heat change during the reaction was detected and recorded. All titrations were repeated in triplicate. The reaction kinetics parameters were calculated using the Origin software package [33] and are described in the Supplementary Method section (S1) [34].

Determination of protein secondary structure

Far-UV CD spectra of purified MmuM and site-directed mutants were recorded at 25°C (Aviv 202 CD Spectrometer). Proteins were dialysed against 150 mM NaCl, 5 mM ZnCl2, 1 mM βME and 20 mM Tris/HCl, pH 7.5, for 4 h after immobilized metal affinity chromatography purification and then dialysed against 100 mM NaCl, 100 mM KCl and 20 mM Tris/HCl, pH 7.5, and diluted to a concentration of 0.3 mg·ml−1 for CD spectrometry. Samples were placed in a 0.1 cm path-length cuvette with ellipticities (θ) recorded at a wavelength range of 200–250 nm (Supplementary Figure S2).

RESULTS AND DISCUSSION

Biological functions and distribution of HMT

As noted above, MmuM enables certain E. coli strains [35] to utilize the plant compound SMM as a methyl donor in methionine biosynthesis. Most E. coli strains also possess two additional MSs: MetE and MetH, that catalyse the S-methylation of Hcy using 5-MTHF as a methyl donor. MetE is an 85-kDa monomer with a zinc cofactor [36], whereas MetH is a 136-kDa monomer containing zinc and cobalamin cofactors [37]. The E. coli KL19 ΔmetE ΔmetH strain but not the ΔmetE ΔmetH ΔmmuM strain can utilize SMM as a Met source [19], indicating that SMM is a biologically relevant methyl donor. Additionally, the ΔmmuM ΔmetK mutant in E. coli K-12 ilvA pSAMT (carrying the AdoMet transporter gene from Rickettsia prowazekii) requires more Met for cell growth than the ΔmetK (AdoMet synthetase) single mutant grown in the presence of equal AdoMet [38]. This observation suggests that AdoMet is a methyl donor for MmuM in vivo. Besides E. coli, the mmuM gene is present in several clinical pathogens, in most cases encoded on the chromosome. Several mmuM genes are, however, found encoded on plasmids, increasing the possibility of horizontal transfer and dissemination of mmuM in bacteria. Interestingly, humans and other mammals also have a MmuM-like enzyme with activity against SMM and, to a much lesser extent, (S,S)-AdoMet [39]. This enzyme (hBHMT2) is annotated as a member of the BHMT family, not the HMT family, although it does not use betaine as methyl donor [40,41].

Overall structure of MmuM

Purified recombinant MmuM has a molecular mass of approximately 35 kDa as estimated from SDS/PAGE and a calculated molecular mass of 34.49 kDa. The protein crystallized in the space group I222 or P21212 and the structures were determined using the deposited structure of T. maritima MS (PDB entry 1Q7M, N-terminal domain) as an initial molecular replacement search model. MmuM crystals were identified in the oxidized (cysteine disulfide), apo (Zn2+-free) or metallated (Zn2+-Hcy) forms. The final co-ordinates were refined to between ∼1.8 and 2.9 Å resolution with one or two monomers of MmuM present in the asymmetric unit (Figure 2; Table 1). Crystallographic packing analysis using the PISA server [42], along with the size-exclusion chromatography elution profile (Figure 2D), are both consistent with MmuM being a monomer in solution. The resolved metallated and oxidized structures in the space group I222 contain ordered residues 4–259 and 276–310. The missing residues (260–275) constitute a loop region between β7 and α7 that is presumed to be disordered. The apo form, P21212, crystals have three additional disordered regions (34–39, 72–78 and 127–138, Supplementary Figure S3A). The overall crystallographic packing between I222 and P21212 is similar. MmuM monomers have electrostatic interactions between α2'/α1 and the neighbouring α4 region. Relative to the I222 structures, the second monomer in space group P21212 has the region containing α1, α2 and α8 shifted ∼3.0 Å relative to other monomers (Supplementary Figure S3B).

Overall structure of E. coli MmuM

Figure 2
Overall structure of E. coli MmuM

(A) Side and (B) top view of overall structure of MmuM in the form of a (α/β)8 TIM barrel; (C) topological diagram of the MmuM monomer. Missing loop (260–275) is boxed in cyan. (D) Size-exclusion chromatography of purified MmuM, suggesting that the protein is monomeric in solution. Running positions of 66- and 29-kDa molecular mass standards are indicated.

Figure 2
Overall structure of E. coli MmuM

(A) Side and (B) top view of overall structure of MmuM in the form of a (α/β)8 TIM barrel; (C) topological diagram of the MmuM monomer. Missing loop (260–275) is boxed in cyan. (D) Size-exclusion chromatography of purified MmuM, suggesting that the protein is monomeric in solution. Running positions of 66- and 29-kDa molecular mass standards are indicated.

MmuM forms a globular compact structure composed of an (α/β)8 TIM barrel [43], with eight outside α-helices and eight inside parallel β-strands that alternate along the peptide backbone (Figure 2). The protein sequence starts from the α'1 helix, located at the base of the (α/β) barrel. A missing loop (260–274) is located on the outer surface of the protein, opposite the metal/substrate-binding site. Two short helixes α'2/α'3 between β1 and α1 are located on the top of the (α/β) barrel covering the β1, β2 and α1 region, whereas α'5/α'6 (inserted between β3/α3) cover the top of β3–β5.

Structural homology searches (DALI server) [44] reveal that MmuM shares the highest structural similarity (Z-score of 33.6) with the Hcy-binding domain of T. maritima MS (MetH-Tm; PDB entry 1Q7Z) [21]. MetH-Tm contains two domains, the N-terminal Hcy-binding domain and the C-terminal 5-MTHF-binding domain, linked by a flexible loop of ∼12 amino acids. The RMSD value is 2.0 Å for 263 aligned Cα atoms between the N-terminal domain of the MetH-Tm and the MmuM monomer. MmuM also shares a high structural similarity with BMHT from rat liver (rBHMT, Z-score of 27.7, PDB entry 1UMY) with an RMSD value of 2.4 Å for 261 aligned Cα atoms between the N-terminal domain of the rBHMT and MmuM monomers. rBHMT contains an extended ∼30-residue C-terminal region, which ends as a long α-helix that is involved in assembly of the active tetramer [20,45]. HMT has an elongated α2 and an additional helix–turn–helix at α'5 and α'6, whereas BHMT and MS have a much shorter β3-α3 loop (Figure 3). All three of these proteins have a conserved cysteine on β6 and a cysteine–cysteine group at β8. These three cysteines are metal-binding and form the enzyme active site.

MmuM belongs to the HMT superfamily of proteins

Figure 3
MmuM belongs to the HMT superfamily of proteins

Sequence alignments (ClustalW2) of MmuM from E. coli with HMTs from Bacillus subtilis (BSHMT) and Arabidopsis thaliana (ATHMT1), with MS from T. maritima (MetH-Tm) and with BHMT from rat liver (rBHMT). Zn2+-binding cysteine residues are labelled with *. Tyr71 and Thr169 (▲) are critical for MmuM activity. MmuM missing loop (260–275) is in a solid box. Unique α'5/ α'6 loop region of HMTs are in a dashed box.

Figure 3
MmuM belongs to the HMT superfamily of proteins

Sequence alignments (ClustalW2) of MmuM from E. coli with HMTs from Bacillus subtilis (BSHMT) and Arabidopsis thaliana (ATHMT1), with MS from T. maritima (MetH-Tm) and with BHMT from rat liver (rBHMT). Zn2+-binding cysteine residues are labelled with *. Tyr71 and Thr169 (▲) are critical for MmuM activity. MmuM missing loop (260–275) is in a solid box. Unique α'5/ α'6 loop region of HMTs are in a dashed box.

The Zn2+-binding active site

MmuM requires Zn2+ for enzymatic activity [14]. The oxidized MmuM structure is in a metal-free state with a disulfide bridge between Cys229 and Cys295 (Figure 4A). The biological role, if any, of oxidized form MmuM is not known. However, the oxidized protein can be reversibly reduced and charged with zinc to generate the metallated form. The MmuM apo form (also Zn2+ free) has Cys229 and Cys295 in the reduced thiol form with a sulfur-to-sulfur distance of 4.3 Å (Figure 4B). This suggests that the observed apo form is in an intermediate state between the oxidized and metallated forms. In contrast, the metallated form has Cys229, Cys295 and Cys296 co-ordinated to a Zn2+ centre with distances of 2.4, 2.1 and 3.2 Å respectively (Figure 4C). The co-ordination of Hcy to metal with a distance of 2.8 Å contributes to a near-ideal Zn2+-centred tetrahedron geometry. Additionally, Cys296 in the metallated form appears to form a (partial occupancy) disulfide bond with a molecule of 2-mercaptoethanol, βME (Figure 4C). βME was introduced into the system during the protein dialysis step incorporating Zn2+ into the protein. The oxidized and metallated forms are similar, with an RMSD of 0.28 Å. In contrast, the crystals of apo MmuM are in a different space group, with a RMSD of 0.42 Å as compared with the metallated form. With the absence of the Zn2+ centre or the disulfide covalent linkage, several disordered loop regions are located on the top of the TIM barrel, including the loop region between β2 and α2 surrounding the active-site pocket. This observation suggests that the Zn2+ centre plays an important structural role in addition to being a key player in catalysis.

The Zn2+ binding active site of MmuM

Figure 4
The Zn2+ binding active site of MmuM

The structure of MmuM showing the Zn2+-binding pocket in (A) oxidized, (B) apo and (C) metallated forms; (D) Zn2+-binding site in rBHMT with thiolate ligands and Tyr160, critical for enzyme activity; and (E) MetH-Tm with bound Cd2+ (mimicking Zn2+) and substrate Hcy. 2mFo–DFc maps are shown at a contour level of 1.5 σ.

Figure 4
The Zn2+ binding active site of MmuM

The structure of MmuM showing the Zn2+-binding pocket in (A) oxidized, (B) apo and (C) metallated forms; (D) Zn2+-binding site in rBHMT with thiolate ligands and Tyr160, critical for enzyme activity; and (E) MetH-Tm with bound Cd2+ (mimicking Zn2+) and substrate Hcy. 2mFo–DFc maps are shown at a contour level of 1.5 σ.

Compared with other similar structures, MmuM has a unique Zn2+-binding pocket. In the structure of rBHMT, the Zn2+ centre is co-ordinated with three cysteine residues and Tyr160 on β4 as the fourth ligand (Figure 4D). A tyrosine at this position is a hallmark of the BHMT family. Mutagenesis studies have confirmed the key role of this tyrosine, as none of the mutants (Y160A, Y160F or Y160T) has significant BHMT activity [45]. HMT, as well as MS family proteins, have a conserved threonine (Thr169) instead of tyrosine at the corresponding position on β4. MmuM does have a tyrosine (Tyr71) close to the Zn2+-binding site from β2. Tyr71 is highly conserved in the HMT family, but is replaced by phenylalanine in the MS family of proteins.

The co-substrate Hcy binds the Zn2+ centre at a similar position in MmuM and MetH-Tm (Figure 4E) and is sandwiched between a conserved phenylalanine (MetH-Tm) or tyrosine (MmuM) and Zn2+. To probe the role and function of the Tyr71 near the metal-binding site, sets of site-directed mutants were designed and assayed. Y71F mutant MmuM has decreased activity against co-substrate SMM, but retains a Vmax ∼ 10% of that measured of the WT form (Supplementary Figure S1B; Supplementary Table S2). Meanwhile, the Y71A mutant is >20-fold less active compared with WT. This suggests that loss of the large aromatic side chain significantly interferes with substrate recognition and interaction. Interestingly, the Y71T mutant has no detectable activity. As all the MmuM mutants retain similar secondary structure elements to WT protein (Supplementary Figure S2), a possible explanation is that the threonine hydroxy group provides a hydrogen-bonding interaction, forcing the substrate Hcy away from its active conformation. To gain further insight into the differences between the HMT and BHMT active-site arrangements, a double mutant containing Y71F and T169Y was designed to better mimic the BHMT substrate-binding pocket; this mutant displayed no activity however. These data indicate that Tyr169 in MmuM T169Y mutant interacts with Zn2+ in much the same way as Tyr160 does in rBHMT (Figure 4D) [45].

Modelling MmuM substrate recognition and binding

The catalytic mechanism of BHMT has been dissected, demonstrating that the reaction may proceed through a transition state where the activated methyl group reacts directly with the homocysteine thiol group [46]. HMT is both structurally and functionally similar to BHMT and probably retains a similar transition state of the methyl transfer chemistry. The structure of metallated MmuM has the co-substrate Hcy bound to the Zn2+ centre, but the position of the other co-substrate, the methyl donor, was not apparent from any of our attempts at co-crystallization. The various enzymes that methylate Hcy use diverse methyl donors: MS uses 5-MTHF and BHMT uses betaine, whereas the HMT family proteins generally utilize SMM, (R,S)-AdoMet, or both. Previously, we demonstrated that MmuM can use SMM or (R,S)-AdoMet as the methyl donor, but not (S,S)-AdoMet or S-ribosylMet [14]. To understand the basis for the observed substrate preference, and the reaction mechanism, co-substrates SMM and (R,S)-AdoMet and substrate mimics (S,S)-AdoMet and S-ribosylMet were docked into the metallated form of MmuM to detail the protein–substrate interaction (Supplementary Table S1; Figure 5).

MmuM substrate binding and recognition

Figure 5
MmuM substrate binding and recognition

(A) Modelling of different methyl donors with metallated form protein. Bound Hcy (green) locates on the top of the pocket. (A) Modelled (R,S)-AdoMet probed near Hcy; (B) detailed interactions between (R,S)-AdoMet and Hcy. The S-methyl group in (R,S)-AdoMet is close to the Hcy thiol group, facilitating the methyl transfer. (C) Modelled (S,S)-AdoMet; (D) (S,S)-S-ribosylMet; (E) (R,S)-S-ribosylMet and (F) SMM near the active site respectively.

Figure 5
MmuM substrate binding and recognition

(A) Modelling of different methyl donors with metallated form protein. Bound Hcy (green) locates on the top of the pocket. (A) Modelled (R,S)-AdoMet probed near Hcy; (B) detailed interactions between (R,S)-AdoMet and Hcy. The S-methyl group in (R,S)-AdoMet is close to the Hcy thiol group, facilitating the methyl transfer. (C) Modelled (S,S)-AdoMet; (D) (S,S)-S-ribosylMet; (E) (R,S)-S-ribosylMet and (F) SMM near the active site respectively.

(R,S)-AdoMet has a good docking contrast with metallated MmuM, the best estimated inhibition constant being 1.9 μM. The aromatic adenine ring of (R,S)-AdoMet lies in a pronounced hydrophobic binding pocket with the exocyclic amino group interacting with the backbone of Gly294 and Asp20 on β1. The α-amino group of (R,S)-AdoMet is stabilized through an electrostatic interaction with Glu134, whereas the α-carboxy group interacts with Gln72 on β2 through a hydrogen bond (Figures 5A and 5B). Gln72 and Glu134 could be the key factors determining HMT's preference for SMM or (R,S)-AdoMet. Glu134 is located on the α'5/α'6 region connecting β3 and α3. The α'5/'6 region lies on the protein surface and is a unique feature of HMT family proteins. This region is disordered in our apo MmuM structure, indicating possible flexibility as a role in protein–co-substrate interaction. Gln72 is located on the β2/α2 loop (71–79, L2) region, which is also flexible in the MmuM apo form. L2 is critical for the substrate binding in BHMT. rBHMT is very sensitive to cleavage between rArg86 and rLys93, but has substantial resistance to proteolysis when bound to the Hcy mimic S-(δ-carboxybutyl)-homocysteine [45]. Gln72 in MmuM is a tyrosine in rBHMT (rTyr77), and this residue plays a role in Hcy recognition [47]. Gln72 in MmuM opens a larger pocket, allowing the binding of co-substrate (R,S)-AdoMet.

Compared with (R,S)-AdoMet, the ‘undamaged’ (S,S)-AdoMet binds metallated MmuM with a very similar estimated binding free energy and inhibition constant (Supplementary Table S1). Most of the protein–ligand interactions remain the same; however, there is a difference between the orientations of the S-methyl positions (Figure 5C). Although the S-methyl group in (R,S)-AdoMet faces the bound Hcy in a predicted catalytically productive mode (Figure 5B), in the (S,S)-AdoMet model, the activated methyl group faces outwards, pointing in the opposite direction to the thiol group of the Hcy co-substrate. As a consequence, (S,S)-AdoMet is predicted to bind but not to serve as a methyl donor.

Additionally, modelling of a S-ribosylMet–MmuM interaction does not result in a binding pattern similar to that of either the AdoMet diastereomers. (S,S)-S-ribosylMet docks into the substrate-binding pocket in a non-productive orientation, with the α-amino and carboxy group near the co-substrate Hcy and the ribosyl group on the protein surface (Figure 5D). The interaction has an estimated inhibition constant of 827 μM, which is approximately 500-fold weaker than that for the metallated MmuM–(R,S)-AdoMet interaction. (R,S)-S-ribosylMet binds the protein with an even higher free energy change and a similar geometry (Figure 5E). As S-ribosylMet is not a productive methyl donor for MmuM [14], we propose that the presence of the aromatic adenine ring in AdoMet helps to correctly position the methyl donor and thus to facilitate access of the activated S-methyl group to the Hcy co-substrate.

Although SMM is a good methyl donor in vitro, modelling suggests that it binds MmuM less tightly than the (R,S)-AdoMet diastereomer. In addition, our top three docking solutions place SMM deep in a pocket, in a conformation not productive in terms of methyl transfer (Figure 5F). Additional bond conformations (Figure 5F, rank 4) do show a productive solution with SMM bound in a fashion similar to that of the Met moiety of (R,S)-AdoMet. This suggests that SMM may also access Hcy from α'5/α'6 and L2 region. As SMM is small compared with AdoMet, it is not surprising that docking algorithms predict a relative lower binding affinity. The prediction of a higher binding affinity for (R,S)-AdoMet than for SMM is particularly useful. Difficulties in separating (R,S)-AdoMet and (S,S)-AdoMet along with the spontaneous interconversion of the diastereomers make it difficult to measure kinetic parameters for MmuM with (R,S)-AdoMet as methyl donor [14,18].

Specific potassium ion requirement for MmuM activity

Human BHMT (hBHMT) (PDB entry 4M3P) has a K+ ion near the Zn2+ site that is critical for the catalytic activity [48]. hBHMT possess a much lower enzymatic activity in the K+-deficient betaine–Hcy reaction. By comparison, our kinetic data indicate that the HMT MmuM reaction maintains the same activity with or without K+ (Supplementary Table S2). Interestingly, we did observe a significant unexpected electron density located adjacent to the substrate-binding pocket in the MmuM oxidized form (Figure 5A). This electron density is located near the α'2 helix and closest to NMet23 with distances of 3.3 Å and a contour level of 8 σ in the calculated mFo–DFc map. The density is also observable in the metallated form of the protein, but has a much lower contour level. The position does not exhibit an anomalous signal at 0.9786 Å, ruling out the possibility of Ni2+ or Zn2+ ions. As there is a prominent dipole interaction of helix α'2 (N-terminus) pointing at the feature, we fitted the density as chloride in the deposited structure.

To summarize, we present the first structure of a member of the homocysteine methyltransferase family, a widely distributed enzyme family that plays key roles in the activated methyl cycle. Using a combination of X-ray crystallography, in vitro and in silico assays, the work provides a structural basis for catalysis and substrate discrimination.

AUTHOR CONTRIBUTION

Kunhua Li, Andrew Hanson and Steven Bruner designed the research. Louis Bradbury carried out the initial MmuM cloning and expression. Kunhua Li and Gengnan Li carried out the research. Kunhua Li, Andrew Hanson and Steven Bruner interpreted the results and wrote the paper.

We thank the staff on the LS-CAT 21-ID-F and 21-ID-G beamlines at Argonne National Laboratory, National Synchrotron Light Source, Lemont, IL, U.S.A., for X-ray access and data collection. We thank Professor Stephen J. Hagen for CD spectrometer access and Dr Zhanglong Liu for assistance on CD data collection.

FUNDING

This work was supported by funds from the University of Florida (to S.D.B.); and the U.S. National Science Foundation [grant number MCB-1153413 (to A.D.H.)].

Abbreviations

     
  • 5-MTHF

    5-methyltetrahydrofolate

  •  
  • AdoMet

    S-adenosyl-L-methionine

  •  
  • BHMT

    betaine homocysteine methyltransferase

  •  
  • Hcy

    L-homocysteine

  •  
  • HMT

    homocysteine S-methyltransferase

  •  
  • ITC

    isothermal titration calorimetry

  •  
  • LS-CAT

    Life Sciences Collaborative Access Team

  •  
  • Met

    L-methionine

  •  
  • MMT

    methionine S-methyltransferase

  •  
  • MS

    methionine synthase

  •  
  • SMM

    S-methyl-L-methionine

  •  
  • S-ribosylMet

    S-ribosyl-L-methionine

  •  
  • WT

    wild-type

  •  
  • βME

    2-mercaptoethanol

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Author notes

1

Present address: Department of Biology, York College, City University of New York, New York, NY 11451, U.S.A.

Supplementary data