The Protein Journal

, 27:303

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from Mycoplasma pneumoniae


  • Amber N. Hancock
    • Department of ChemistryVirginia Tech
  • R. Shane Coleman
    • Department of Chemistry and PhysicsRadford University
  • Richard T. Johnson
    • Department of Chemistry and PhysicsRadford University
  • Catherine A. Sarisky
    • Department of ChemistryRoanoke College
    • Department of Chemistry and PhysicsRadford University

DOI: 10.1007/s10930-008-9138-z

Cite this article as:
Hancock, A.N., Coleman, R.S., Johnson, R.T. et al. Protein J (2008) 27: 303. doi:10.1007/s10930-008-9138-z


5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839–843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (Km) for ATP from 76 to 1,200 μM and the Km for 5-formylTHF from 2.5 to 7.1 μM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.


5-FormyltetrahydrofolateSite-directed mutagenesis5,10-Methenyltetrahydrofolate synthetaseATPKinetics







Adenosine diphosphate


Adenosine triphosphate


Circular dichroism spectroscopy


N-Cyclohexyl-2-aminoethanesulfonic acid


Michaelis constant




2-(N-Morpholino)ethanesulfonic acid


5,10-Methenyltetrahydrofolate synthetase


Polymerase chain reaction


Phosphate buffered saline





1 Introduction

Tetrahydrofolates (THFs) are cofactors that donate or accept single carbon units in metabolic processes. These molecules are involved in fundamental cellular reactions such as purine synthesis, thymidylate synthesis, DNA repair, amino acid synthesis, and amino acid degradation [1]. Based on the importance of these reactions, it is not surprising that defects in folate metabolism have been linked to increased rates of cancer [2], heart disease [3], and neural tube defects [4].

The various forms of THF differ in the oxidation state and/or the position of the carbon to be donated. The oxidation state of the donated carbon can range from the equivalent of formate (most oxidized) to the equivalent of methanol (most reduced). 5-Formyltetrahydrofolate (5-formylTHF) is a form of tetrahydrofolate that does not directly act as a carbon donor, but is converted through multiple steps into forms of THF that do donate carbons. 5-FormylTHF is the most stable of the tetrahydrofolates and has been hypothesized to be a storage form for this class of molecules [5]. The only enzyme known to use 5-formylTHF as a substrate is 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC MTHFS catalyzes the conversion of 5-formylTHF into 5,10-methenyltetrahydrofolate (5,10-methenylTHF) coupled to the hydrolysis of ATP to ADP (Fig. 1). The details of this reaction are of particular interest because 5-formylTHF is administered (under the trade name of Leucovorin) in some forms of chemotherapy. In general, 5-formylTHF is either given as a rescue agent following methotrexate treatment or it is co-administered with fluorouracil as an enhancer [1].
Fig. 1

MTHFS catalyzes the conversion of 5-formylTHF to 5,10-methenylTHF coupled to the hydrolysis of ATP

A mechanism for the conversion of 5-formylTHF to 5,10-methenylTHF has been proposed (Fig. 2) [6, 7]. The first step in this mechanism is a nucleophilic attack on the gamma phosphate of ATP by the formyl oxygen of 5-formylTHF, resulting in an iminium phosphate intermediate. The N10 then makes a nucleophilic attack on the formyl carbon to displace the phosphate and form the 5-membered pteridine ring. Results of non-equilibrium isotope exchange studies suggest that the nucleophilic attack by N10 is the rate limiting step in this reaction [6]. Recent research using site-directed mutagenesis and MTHFS inhibitors has indicated the importance of an active site tyrosine in the substrate specificity of this reaction step [8].
Fig. 2

Proposed mechanism for the conversion of 5-formylTHF to 5,10-methenylTHF catalyzed by MTHFS [6, 7]

There are currently available crystal structures of MTHFS from Mycoplasma pneumoniae [9, 10], Bacillus anthracis [11], and Bacillus subtilis (PDB code 1ydm). For M. pneumoniae, a co-crystal structure exists of MTHFS with bound 5-formylTHF, ADP, and free phosphate ([9], PDB code 1U3G). In this structure, a highly conserved arginine at position 115 (R115) is positioned to make hydrogen bonding and ionic contacts with the free phosphate ion (Fig. 3). This is consistent with a mechanism in which an iminium phosphate intermediate is formed. The positively charged side chain of arginine is positioned to draw electron density away from the phosphate in either its ATP or iminium intermediate form. This would result in a more electrophilic phosphorus or iminium carbon and facilitate nucleophilic attack at these positions (Fig. 2, steps 2 and 3, respectively).
Fig. 3

Graphic based on the co-crystal structure of MTHFS [9] showing 5-formylTHF, ADP, phosphate, R115, and K120. Figure prepared with the program MOLMOL [12]

The enzyme also contains a highly conserved lysine at position 120 (K120). In the co-crystal structure, the side chain of this amino acid is oriented away from the substrates and active site of the enzyme (Fig. 3). Although K120 is highly conserved, the side chain does not appear to interact directly with the substrates based on the crystal structure. However, the backbone amide associated with this residue is in position to make a hydrogen bond with ATP [9].

While the contacts observed in the co-crystal structure of MTHFS support the formation of a phosphorylated THF intermediate, no crystal structure of this intermediate with the enzyme currently exists. To further validate the contacts made between R115 and a potential phosphorylated THF intermediate, we have created and characterized a mutant MTHFS in which the arginine has been replaced with alanine (R115A) through site-directed mutagenesis. We have investigated the role of the lysine at position 120 as well through a similar mutation replacing this amino acid with alanine (K120A). These residues are likely to be important, either directly or structurally, in the mechanism of reaction as both are highly conserved across several species [9].

2 Materials and Methods

2.1 Materials

2-(N-Morpholino)ethanesulfonic acid (MES) and [6R,6S]-5-formylTHF (as folinic acid calcium salt pentahydrate) were purchased from Acros Organics. Sodium chloride, sodium phosphate, imidazole, phosphate buffered saline (PBS), magnesium chloride, Triton X-100, 2-mercaptoethanol, kanomycin monosulfate, and ATP disodium trihydrate were purchased from Fisher Scientific. Spectinomycin dihydrochloride was purchased from Sigma. Desalted oligonucleotides were purchased from the DNA Oligonucleotide Synthesis Core at Massachusetts General Hospital. Pfu polymerase was purchased from Stratagene. The Sequenase™ 7-deaza-dGTP DNA Sequencing Kit was purchased from USB Corporation. Talon Metal Affinity Resin was purchased from Clontech Laboratories, Inc. Sephadex G-25 resin was purchased from Amersham Biosciences. A plasmid containing the wild type gene for M. pneumoniae MTHFS fused to a poly-histidine tag (Chen et al. 2004) was a generous gift from Dr. Sung-Hou Kim as was BL21(DE3)/pSJS1244 [13], an expression cell line for the protein.

2.2 Site-directed Mutagenesis

The codons at positions 115 and 120 of wild type MTHFS were changed from arginine to alanine or lysine to alanine, respectively, using a modification of the protocol for the Stratagene QuikChange® Site-Directed Mutagenesis Kit. The primers were desalted and used without further purification. The forward and reverse primers used for the R115A mutant were as follows. Forward: 5′-G-GTA-GGC-TTT-AAT-AAA-GAC-AAT-TAC-GCT-CTA-GGC-TTT-GGC-AAG-GGC-3′; Reverse: GCC-CTT-GCC-AAA-GCC-TAG-AGC-GTA-ATT-GTC-TTT-ATT-AAA-GCC-TAC-C-3′ where the nucleotide substitutions are bolded and underlined. The primers used for the K120A mutant were as follows. Forward: 5′-C-CGT-CTA-GGC-TTT-GGC-GCG-GGC-TAT-TAT-GAC-CGT-TAT-TTA-ATG-C-3′; Reverse: 5′-G-CAT-TAA-ATA-ACG-GTC-ATA-ATA-GCC-CGC-GCC-AAA-GCC-TAG-ACG-G-3′. 2.5 units of Pfu DNA polymerase was added to 50 μL of solution containing 125 ng of each primer, 25 ng of template plasmid, and 50 μM of each dNTP. This solution was subjected to 16 cycles of PCR and then placed on ice. Ten units of DpnI were added and the reaction mixture was incubated at 37 °C for 1 h. Two microliters of the resulting mix were used to transform XL1-Blue competent cells using the method of Inoue [14]. Transformed cells were grown overnight in Luria-Bertani (LB) medium in the presence of 50 μg/mL kanomycin. Plasmid DNA was extracted via a Qiagen Miniprep Kit following the manufacturer’s instructions. The DNA was then sequenced using a Sequenase™ 7-deaza-dGTP DNA Sequencing Kit and a dye labeled T7 primer following the manufacturer’s instructions. Sequencing products were resolved using a LI-COR 4300 DNA Analyzer to confirm both mutations.

2.3 Protein Expression and Purification

Plasmids containing the wild type, R115A, and K120A MTHFS genes were transformed into the Escherichia coli strain BL21(DE3)/pSJS1244 using the method of Inoue [14]. Transformed cells were grown in LB medium containing 50 μg/mL kanomycin and 30 μg/mL spectinomycin to an optical density of 0.9 at 600 nm. Protein expression was then induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside for 16 h at room temperature. Cells were pelleted by centrifugation for 10 min at 2,000g at room temperature in a Beckman TJ-6 centrifuge. The supernatant was discarded and the following freeze/thaw method was used to extract the protein from the cells. Each cycle of freeze/thaw involved placing the cell pellets in liquid nitrogen for 5 min followed by incubation on ice until pellets were free flowing. After three cycles of freeze/thaw, cell debris was resuspended in 1 mL of PBS and gently shaken on ice for 1 h. Cell debris was then pelleted for 2 min at 16,100g in an eppendorf 5415 D centrifuge. MTHFS was purified from the supernatant using Talon metal affinity resin following the manufacturer’s instructions. The elution of MTHFS from the resin was accomplished with 300 mM imidazole, which was removed through size exclusion chromatography with Sephadex G-25 resin. Eluted MTHFS was quantitated by UV/visible spectroscopy on Hewlett Packard 8452A spectrophotometer (ε280 = 14,650 cm−1 M−1) and stored in 50 mM HEPES pH 7.0, 30 mM NaCl, 0.1% Triton X-100, 0.01% 2-mecaptoethanol at 4 °C. Preparations were observed to be pure when analyzed by polyacrylamide gel electrophoresis with a Coomassie blue stain [15]. For the protein samples used in circular dichroism analysis, the storage buffer was exchanged for 10 mM phosphate (pH 7.0) using two rounds of size exclusion chromatography.

2.4 Kinetic Assays

Kinetic data were collected using a modification of a previously published method [16]. The production of 5,10 methenylTHF was monitored at 360 nm (ε360 = 25,100 M−1 cm−1). The standard kinetic assay solution contained 200 mM MES pH 6.0, 30 mM sodium chloride, 10 mM magnesium chloride, 0.1% Triton X-100, 0.01% 2-mercaptoethanol, 200 μM 5-formylTHF, 1 mM ATP, and 2 μg of MTHFS. Ten micrograms of enzyme were used for the direct comparisons of wild type MTHFS and the R115A mutant depicted in Fig. 4. For determinations of Km, substrate concentration ranges were 25–4,000 μM ATP and 0.5–32 μM 5-formylTHF depending on the mutant enzyme investigated. MTHFS was added to a solution containing all other reagents at 37 °C, thoroughly mixed, and the absorbance at 360 nm measured every 5 s between 0.5 and 2 min on a Hewlett Packard 8452A spectrophotometer. One centimetre path length cells were used for the ATP measurements and 10 cm path length cells were used for the 5-formylTHF measurements due to a need for greater sensitivity. The slopes of the resulting data were used to calculate initial rates. Kinetic parameters were determined using an Eadie-Hofstee plot [17].
Fig. 4

Comparison of the activities of wild type MTHFS (■) with the R115A mutant (♦). Concentrations of the product 5,10-methenyltetrahydrofolate were calculated from the absorbance at 360 nm. No activity for the R115A mutant was observed after 30 min of incubation. Ten micrograms of each enzyme were used for this comparison

2.5 Circular Dichroism Spectroscopy

Data were collected on an Olis DSM-10 Circular Dichroism Spectrophotometer every 0.5 nm between 200 and 250 nm with a slit width of 0.5 mm. Protein concentrations were 0.1 mg/mL for each sample measured 0.2 cm rectangular cells. Measurements were made at 24 °C. Background and sample spectra were taken in triplicate and smoothed using the Savitzky-Golay method [18]. Smoothed spectra were averaged and the average background subtracted from the average sample spectra. The CDPro software package [19] was used to determine the fraction of alpha-helix, beta-sheet, and turn in the wild type and each mutant. CD data (every nanometer between 200 and 240 nm) were analyzed using the SDP48 basis set with three methods: CDSSTR [19, 20], CONTIN/LL [19, 21], and SELCON3 [20, 22].

3 Results and Discussion

3.1 Role of R115

5-FormylTHF has been proposed to go through an iminium phosphate intermediate in its conversion to 5,10-methenylTHF by MTHFS [6, 7] (Fig. 2). In the co-crystal structure with bound 5-formylTHF, ADP, and phosphate, the phosphate is located within 3.50 Å of the formyl group. This places it in position to accept a nucleophilic attack from the oxygen on the formyl group. The η1 and η2 nitrogens of R115 are within hydrogen bonding distance (3.33 Å) of the oxygens on the phosphate. It is worthy of note that while multiple backbone amides are within hydrogen bonding distance of the phosphate, R115 is only side chain close enough to form these contacts in the crystal structure. The positive charge on this side chain is aptly positioned to facilitate the reaction by making the phosphorus (step 2) and/or the iminium carbon (step 3) more electrophilic. If the mechanism detailed in Fig. 2 is correct, then R115 likely plays an integral role in the catalytic action of MTHFS.

To test the importance R115, we changed this amino acid to alanine using site-directed mutagenesis as described in Sect. 2. Kinetic assays were then run on both the wild type and R115A mutant enzymes. The results of these assays are shown in Fig. 4. Replacement of arginine with alanine at position 115 has substantially reduced the activity of the enzyme. No activity was detected from the R115A mutant even after 30 min of incubation (data not shown).

The following precautions were taken to rule out the lack of activity of the R115A mutant being due to artifacts in preparation and purification. The genes for both wild type and R115A mutants were verified by DNA sequencing. Two preparations of the R115A mutant were tested for catalytic activity. All other site-directed mutants of MTHFS that we have produced to date have demonstrated at least limited catalytic activity. These mutants included amino acids inside and outside the active site of the enzyme. Other positions tested were R14, Y122, Y123 and Y125 (data not shown). For all these mutations, the wild type amino acid was replaced with alanine.

Multiple explanations can be given for the loss in activity of the R115A mutant. One explanation is that replacement of the arginine with alanine may have removed a critical charge–charge interaction with the iminium phosphate intermediate in the proposed mechanism. Another explanation is that the arginine at position 115 was critical for correct folding of the enzyme. To rule out large perturbations to the enzyme’s structure, circular dichroism (CD) spectroscopy was carried out on the wild type and the R115A mutant as described in Sect. 2 The results (Fig. 5) are consistent with both the wild type, the R115A mutant, and the K120A mutant (discussed below) being folded, indicating that the mutations have not prevented the folding of MTHFS. The fraction of alpha-helix, beta-sheet, and turn in the wild type and each mutant was calculated using the methods CDSSTR [19, 20], CONTIN/LL [19, 21], and SELCON3 [20, 22]. While there was intra-method agreement in the results for the wild type and the two mutant enzymes, there was poor agreement between the methods. This lack of agreement prevents a more detailed discussion of the CD data. More subtle differences between the folded structures of the wild type and mutant enzymes would be best addressed through X-ray crystallography. Still, the CD data are consistent with folded proteins. These data taken together with the lack of activity for the R115A mutant and the position of R115 in the crystal structure support the proposed mechanism for this enzyme.
Fig. 5

Circular dichroism spectra of MTHFS wt protein (grey solid line), R115A mutant (black broken line), and K120A mutant (black solid line)

3.2 Role of K120

In addition to R115, we investigated the role of K120 in the catalytic activity of the enzyme. In the co-crystal structure, the side chain of this amino acid is not positioned to interact with either the 5-formylTHF, the ADP, or the phosphate in the active site of the enzyme. However, replacement of this lysine with alanine weakens the binding of both ATP and 5-formylTHF (Table 1). The Km for 5-formylTHF (7.1 μM) is somewhat higher for K120A than the Km of the wild type (2.5 μM) for this substrate. More substantial is the lower affinity of K120A for ATP. The Km for K120A’s interaction with ATP (1,200 μM) is increased by over an order of magnitude compared to the wild type (76 μM) indicating substantially weaker binding of this substrate. The values for kcat are slightly higher for K120A compared to wild type. However, this difference is small and is likely a result in subtle differences in preparation or quantitation. The loss of affinity of K120A for ATP may be due to protein structural differences resulting from the replacement of the lysine with alanine. Backbone amides at positions 117, 118, 119, and 120 are oriented to make hydrogen bonding contacts with the ADP and phosphate in the co-crystal structure [9]. These backbone amides form a glycine rich loop that is highly conserved across the MTHFS family of proteins [9, 23]. While the loop is highly conserved across all species, the lysine at position 120 in M. pneumoniae is only conserved in bacteria [9]. The loop may be held in proximity to ATP by a contact made between K120 and another amino acid in the protein. This hydrogen bonding partner may be D81. Rotation about χ1 is sufficient to reduce the distance between K120 NZ and D81OE1 to 3.04 Å. This hypothesized contact is further supported by the presence of a highly conserved aspartate or glutamate in the equivalent position of D81 across bacterial MTHFS [9]. Work is presently underway to further characterize this interaction. Thus, loss of the K120 side chain contacts may result in conformational changes in this loop region that remove one or more amide hydrogen bond contacts to ATP.
Table 1

Kinetic parameters for wt and K120A forms of MTHFS

MTHFS form

Km ATP (μM)

kcat ATP (s−1)

Km 5-formylTHF (μM)

kcat 5-formylTHF (s−1)

Wild type

76 ± 9

1.0 ± 0.1

2.5 ± 0.3

0.79 ± 0.04


1,200 ± 2

1.7 ± 0.1

7.1 ± 1.5

1.3 ± 0.2

Kinetic parameters were determined as described in Sect. 2. No activity was detected in the R115 mutant. The ±values represent the standard error

This work supports and further elucidates the consensus on the catalytic details of the conversion of 5-formylTHF to 5,10-methenylTHF by MTHFS. The lack of activity of the R115A mutant is consistent with this arginine making critical contacts to the γ-phosphate of ATP or the iminium phosphate intermediate in the published mechanism [6, 7]. The decrease in affinity of K120A for both substrates, ATP in particular, is consistent with the replaced lysine providing a structural role in holding a loop in place that makes contacts with the substrates. These results provide a greater understanding of the roles of these highly conserved residues and of MTHFS as a whole.


We would like to thank Mark Bouley (Radford Univeristy) for help with purification protocols, Rosalind Kim (Structural Genomics Center) for advice in expression and purification of wild type MTHFS, and Sung-Hou Kim (Structural Genomics Center) for the generous gifts of the plasmid containing the MTHFS gene and the BL21(DE3)/pSJS1244 expression line for the protein. This work was funded through the Jeffress Memorial Trust (J-788) and Radford University internal grants. DNA sequencing was supported through the LI-COR Biosciences Genomics Education Matching Fund Program.

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© Springer Science+Business Media, LLC 2008