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Originally published In Press as doi:10.1074/jbc.M306747200 on August 5, 2003

J. Biol. Chem., Vol. 278, Issue 43, 41789-41797, October 24, 2003
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Catalytic and Thermodynamic Properties of Tetrahydromethanopterin-dependent Serine Hydroxymethyltransferase from Methanococcus jannaschii*

Sebastiana Angelaccio, Roberta Chiaraluce, Valerio Consalvi, Bärbel Buchenau{ddagger}, Laura Giangiacomo, Francesco Bossa§, and Roberto Contestabile

From the Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and §Centro di Eccellenza di Biologia e Medicina Molecolare, Università degli Studi di Roma "La Sapienza," Piazzale Aldo Moro 5, Roma 00185, Italy and the {ddagger}Max-Planck-Institut für Terrestrische Mikrobiologie and Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität, Karl-von-Frisch-Strasse, Marburg D-35043, Germany

Received for publication, June 25, 2003 , and in revised form, August 1, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The reaction catalyzed by serine hydroxymethyltransferase (SHMT), the transfer of C{beta} of serine to tetrahydropteroylglutamate, represents in Eucarya and Eubacteria a major source of one-carbon (C1) units for several essential biosynthetic processes. In many Archaea, C1 units are carried by modified pterin-containing compounds, which, although structurally related to tetrahydropteroylglutamate, play a distinct functional role. Tetrahydromethanopterin, and a few variants of this compound, are the modified folates of methanogenic and sulfate-reducing Archaea. Little information on SHMT from Archaea is available, and the metabolic role of the enzyme in these organisms is not clear. This contribution reports on the purification and characterization of recombinant SHMT from the hyperthermophilic methanogen Methanococcus jannaschii. The enzyme was characterized with respect to its catalytic, spectroscopic, and thermodynamic properties. Tetrahydromethanopterin was found to be the preferential pteridine substrate. Tetrahydropteroylglutamate could also take part in the hydroxymethyltransferase reaction, although with a much lower efficiency. The catalytic features of the enzyme with substrate analogues and in the absence of a pteridine substrate were also very similar to those of SHMT isolated from Eucarya or Eubacteria. On the other hand, the M. jannaschii enzyme showed increased thermoactivity and resistance to denaturating agents with respect to the enzyme purified from mesophilic sources. The results reported suggest that the active site structure and the mechanism of SHMT are conserved in the enzyme from M. jannaschii, which appear to differ only in its ability to bind and use a modified folate as substrate and increased thermal stability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serine hydroxymethyltransferase (SHMT,1 EC 2.1.2.1 [EC] ) catalyzes the reversible transfer of C{beta} of serine to tetrahydropteroylglutamate (H4PteGlu) to form glycine and 5,10-methylene-H4PteGlu. In Eukarya and Eubacteria, H4PteGlu functions as a carrier of C1 units in several oxidation states, which are used in the biosynthesis of important cellular components, such as purines and thymidylate, in the regeneration of methionine from homocysteine or, in acetogenic bacteria, in the synthesis of acetyl-CoA. The reaction catalyzed by SHMT represents in these organisms one of the major loading routes of C1 units onto the folate carrier (1). In methanogens and several other Archaea, C1 fragments from formyl to methyl oxidation levels are carried by tetrahydromethanopterin (H4MPT), a pterin-containing compound involved in methanogenesis. Although H4PteGlu and H4MPT are structurally similar in their pterin-like portion (Fig. 1) and in the role as C1 units carriers, they are functionally distinct. H4MPT does not appear to be suited to most of the biosynthetic functions of H4PteGlu. Moreover, the biosynthetic pathways of the two carriers have few, if any, homologies, suggesting the possibility of separate evolutionary origins (2). In the metabolism of folates, SHMT represents a unique link between Archaea and the rest of living beings, in the sense that, whereas all SHMTs clearly share a common evolutionary origin (3), other enzymes that use H4MPT as cofactor do not show any significant homology to their eukaryotic and eubacterial counterparts (2).



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  		FIG. 1.
Structures of tetrahydropteroylglutamate (a) and tetrahydromethanopterin (b).

 

Although a gene encoding SHMT is present in all archaeal genomes so far sequenced, little information is available on the catalytic properties and metabolic role of the enzyme in these organisms. Modified folates are not commercially available, and this has clearly hindered a satisfactory characterization of archaeal SHMTs. Moreover, the purification of the enzyme from Archaea that thrive in extreme environments is complicated by the difficulty of growing these organisms in a laboratory. Two reports of purified SHMT activity, from Methanobacterium thermoautotrophicum (4), recently renamed Methanothermobacter marburgensis (5), and from Sulfolobus solfataricus (6), with limited structural and functional characterization, have been made. In the first report, the enzyme was proposed to function in vivo in the direction of serine biosynthesis. Importantly, both works provided evidence that SHMT was selective toward the modified folate used by the source organisms: H4MPT for M. marburgensis and sulfopterin for S. solfataricus (2, 7).

This study reports on the purification of recombinant SHMT from Methanococcus jannaschii (mjSHMT) and the characterization of its catalytic and thermodynamic properties. The aim of the research was to assess the extent to which mjSHMT is structurally and mechanistically similar to its prokaryotic and eukaryotic counterparts.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Ingredients for bacterial growth were from Difco. Chemicals used in the purification procedure were from BDH, and DEAE-Sepharose and phenyl-Sepharose were from Amersham Biosciences. Enzymes used for the cloning procedures were purchased from Roche Diagnostics. SHMT from Escherichia coli (eSHMT) was expressed and purified as described previously (8). The oligonucleotides used in the PCR reaction were from MWG Biotech. (6S)-H4PteGlu was a gift from A. G. Eprova, Schaffhausen, Switzerland. All other reagents were from Sigma-Aldrich. The bifunctional enzyme methylene-H4PteGlu dehydrogenase/methenyl-H4PteGlu cyclohydrolase from E. coli was purified from a recombinant E. coli bacterial strain kindly provided by Dr. Martino di Salvo (Università "La Sapienza," Rome, Italy) following the procedure described previously (9). NADP-dependent methylene-H4MPT dehydrogenase from Methylobacterium extorquens AM1 and H4MPT purified from M. marburgensis were kindly provided by Rolf Thauer (Max-Planck-Institut für terrestrische Mikrobiologie, Marburg, Germany). The thermostable alcohol dehydrogenase from Thermoanaerobium brockii employed in the threonine aldolase assay was from Sigma-Aldrich.

Cloning of the glyA Gene from M. jannaschii—A pUC18 plasmid with a 1686-bp insert containing the gene encoding mjSHMT was purchased from the American Type Culture Collection. This plasmid (AMJK15) was used as template in a PCR amplification that used 5'-GGGATACATATGGAATATTCGG-3' (upstream) and 5'-GCGAATTCTTAATAGAATCTTAG-3' (downstream) oligonucleotides as primers. NdeI and EcoRI restriction sites were introduced in the primers to insert the amplified 1287-bp DNA fragment into a pET22b expression plasmid (Novagen), which was used to transform E. coli HMS174 (DE3) cells (Novagen). The nucleotide sequence of the insert was determined to confirm that no mismatching had occurred during the PCR amplification.

Expression and Purification—A 100-ml overnight culture of E. coli HMS174 (DE3) cells, transformed with the mjSHMT overexpressing plasmid, was inoculated into 3 liters of Luria-Bertani broth containing ampicillin (100 mg/liter) and grown aerobically at 37 °C to exponential phase (A600 = 0.3–0.4). Expression of mjSHMT was then induced with 0.2 mM isopropyl thio-{beta}-D-galactopyranoside. After 20 h, cells were harvested and suspended in 100 ml of 50 mM Tris/HCl, 1 mM EDTA, pH 7.6. Cell lysis was obtained by the addition of 1 mg of lysozyme per g of packed bacterial cells. After incubation at 25 °C for 30 min, the cells were frozen overnight at –20 °C. After thawing, streptomycin sulfate (10 g/liter) was added to precipitate DNA. The cell extract was then centrifuged at 15,000 x g for 30 min, and the pellet was discarded. Solid ammonium sulfate was added to the yellow supernatant to 60% saturation. The solution was centrifuged at 15,000 x g for 20 min, and the pellet was discarded. The supernatant was directly loaded onto a phenyl-Sepharose column (5 x 15 cm) pre-equilibrated with 50 mM potassium phosphate buffer, pH 7.2, containing ammonium sulfate to 30% saturation. The column was washed with 100 ml of the same buffer, and the protein was eluted using a 600-ml linear gradient from the equilibrating buffer to 20 mM potassium phosphate, pH 7.2. Fractions containing SHMT, detected spectrophotometrically, pooled, and concentrated using centrifuge concentrators (30 kDa cut-off; Vivaspin, Sartorius) to a volume of about 20 ml, were heat-treated for 15 min at 75 °C to precipitate most of the host cell proteins. After centrifugation at 15,000 x g for 30 min, the supernatant containing mjSHMT and a few other proteins was loaded onto a DEAE-Sepharose column (3 x 10 cm) that had been equilibrated with 20 mM potassium phosphate, pH 7.2. The column was washed with 100 ml of the same buffer, and the protein was then eluted with a 500-ml linear gradient of 0–0.4 M NaCl in 20 mM phosphate buffer, pH 7.2. Fractions were pooled and concentrated as in the previous step. The purity of the mjSHMT sample was checked by SDS-PAGE and judged to be >=98%. The protein concentration was determined measuring the absorbance at 280 nm and using a {epsilon}280 = 34990 M–1 cm–1, calculated according to Ref. 10.

Molecular Mass Estimation by Size-exclusion Chromatography—The molecular mass of the native enzyme was estimated by size-exclusion chromatography at 20 °C on a Superose 12 column (Amersham Biosciences), eluted at a flow rate of 0.4 ml/min with 20 mM sodium phosphate, pH 7.2, containing 0.15 M NaCl, controlled by a Dionex pump. Elution was monitored at 280 and 226 nm. The Superose column was calibrated with horse spleen apoferritin (440 kDa, elution volume Ve = 8.0 ml), catalase (232 kDa, Ve = 9.1 ml), rabbit muscle aldolase (158 kDa, Ve = 9.3 ml), BSA (67 kDa, Ve = 9.8 ml), egg albumin (43 kDa, Ve = 10.4 ml), chymotrypsin (25 kDa, Ve = 12 ml), and ribonuclease (13.7 kDa, Ve = 12.5 ml). Ve of the purified SHMT was 9.6 ml.

Molecular Mass Estimation by Analytical Ultracentrifugation—All experiments were conducted at 20 °C on a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics. The protein concentration was in the range of 0.2 to 1.0 mg/ml. Sedimentation velocity experiments were done at 40,000 rpm. Data were collected at 280 nm at a spacing of 0.005 cm with three averages in a continuous scan mode and were analyzed with the program Sedfit (11). Sedimentation coefficients were corrected to s20,w using standard procedures. Sedimentation equilibrium experiments were performed at 16,000, 19,000 and 22,000 rpm. Data were collected at 280 nm at a spacing of 0.001 cm with 10 averages in a step scan mode. Establishment of equilibrium was checked by comparing scans up to 24 h. Data sets were edited with REEDIT (J. Lary, National Analytical Ultracentrifugation Center, Storrs, CT) and fit with NONLIN (PC version provided by E. Braswell, National Analytical Ultracentrifugation Center, Storrs, CT) (12). Data from different speeds were combined for global fitting. Fits to a single species give a Z-average molecular weight. For fits to a monomer-dimer association scheme, the monomer molecular weight was fixed at the value determined from the amino acid sequence. The experiments were performed in 20 mM phosphate buffer, pH 7.2, and in 20 mM sodium formate, pH 3.0, containing 200 µM DTT and 100 µM EDTA.

Kinetic Studies—With serine and H4MPT as substrates, the rate of 5,10-methylene-H4MPT production was determined by oxidizing this compound to 5,10-methenyl-H4MPT, using NADP+ and methylene-H4MPT dehydrogenase from M. extorquens AM1. The initial rate of the reaction, carried out in 75 mM potassium phosphate, pH 7.4, at 37 °C, was calculated from the absorbance change at 340 nm because of NADPH and methenyl-H4MPT formation, using a value of {epsilon}340 = 27 000 M–1 cm–1 (13). The same procedure was employed when H4PteGlu was the pteridine substrate, except that the coupled enzyme employed in the assay was the bifunctional methylene-H4PteGlu dehydrogenase/methenyl-H4PteGlu cyclohydrolase from E. coli, and the {epsilon}340 used in the calculation was 7200 M–1 cm–1 (8). The reaction mixture included mjSHMT at either 0.1 µM, when H4MPT was the pteridine substrate, or 14 µM, when H4PteGlu was used. Kinetic parameters were determined either under anaerobic conditions (N2 pressure equal to 0.3 bar) or in the presence of air, varying the concentration of one substrate while the second was kept constant. The dependence of the initial velocity of the reaction on H4MPT concentration was determined maintaining L-serine at 30 mM and varying H4PteGlu concentration between 0.162 and 0.45 mM.H4MPT was 0.162 mM when serine was varied between 0.09 and 30 mM. The formaldehyde was measured mixing aliquots of the reaction mixture, taken at time intervals, with an equal volume of 0.2% (w/v) 2,4-dinitrophenyl hydrazine in 1 M HCl. The resulting colored solution was diluted in 1 M HCl and used to measure the increase in absorbance at 380 nm because of the formation of a dinitrophenyl hydrazone derivative. For this derivative, a calibration curve, obtained previously using commercial formaldehyde, gave a {epsilon}380 = 57120 M–1 cm–1.

Retroaldol cleavage reactions were carried out in 20 mM potassium phosphate, pH 7.2. The rate of allo-threonine cleavage was measured by coupling the reaction with reduction of the product acetaldehyde by NADH and thermostable alcohol dehydrogenase from T. brockii (14). The rate of the reaction was calculated from the rate of decrease in absorbance at 340 nm, using a value of {epsilon}340 = 6220 M–1 cm–1. Benzaldehyde production from phenylserine cleavage was measured spectrophotometrically at 279 nm, using a molar absorptivity value of {epsilon}279 = 1400 M–1 cm–1 (15). The rate of transamination with both alanine enantiomers was determined according to Ref. 16. Kinetic data analysis, curve-fitting procedures, and statistical analysis were performed using the data manipulation software of Scientist (Micromath, Salt Lake City, UT).

Spectroscopic Techniques—Fluorescence emission measurements were carried out at 20 °C with a LS50B spectrofluorimeter (PerkinElmer Life Sciences) using a 1-cm path length quartz cuvette. Intrinsic fluorescence emission spectra were recorded from 300–450 nm (1-nm sampling interval) with the excitation wavelength set at 295 nm. PLP emission fluorescence was monitored between 330 and 600 nm exciting at 285, 320, and 418 nm.

CD spectra were recorded at 20 °C in a Jasco J-720 spectropolarimeter. Far-UV-CD spectra (190–250 nm) were measured in a 0.1-cm path length quartz cuvette, and near-UV-CD spectra (250–310 nm) and visible-CD spectra (310–500 nm) were measured in a 1.0-cm path length quartz cuvette. The results are expressed as the mean residue ellipticity ([{Theta}]) assuming a mean residue weight of 110 Da per amino acid residue. In all the spectroscopic measurements, 200 µM DTT and 100 µM EDTA were added unless otherwise stated. UV-visible spectra were recorded with a double-beam Lambda 16 PerkinElmer Life Sciences spectrometer equipped with a Peltier thermocontroller set at 20 °C.

Urea-induced Unfolding Equilibrium—Protein samples, at 0.12 mg/ml final concentration, were incubated at 20 °C with increasing concentrations of urea (0–7.9 M) in 20 mM sodium phosphate, pH 7.2, or in 20 mM sodium formate, pH 3.0, in the presence of 200 µM DTT and 100 µM EDTA. After 24 h, a time that was tested to be sufficient to reach equilibrium, far-UV-CD spectra were recorded at 20 °C. To probe the reversibility of the unfolding process, the protein (1.9 mg/ml) was incubated at 20 °C with 7.9 M urea in 20 mM formate, pH 3.0, in the presence of 3.2 mM DTT and 1.6 mM EDTA. After 24 h, the refolding was started by a 16-fold dilution with the same buffer used for the unfolding, containing decreasing denaturant concentrations. The final protein concentration was 0.12 mg/ml. After 2 h, a time that was established to be sufficient to reach equilibrium, far-UV-CD spectra were recorded at 20 °C.

Data Analysis—Far-UV-CD spectra from urea titration were analyzed by the singular value decomposition algorithm (SVD) (17–19) using the software MATLAB (MathWorks, South Natick, MA). SVD is useful to find the number of independent components in a set of spectra and to remove the high-frequency noise and the low-frequency random error. CD spectra in the 210–250-nm region (0.2-nm sampling interval) were placed in a rectangular matrix A of n columns, one column for each spectrum collected in the titration. The A matrix is decomposed by SVD into the product of three matrices: A = U*S*VT, where U and V are orthogonal matrices, and S is a diagonal matrix. The columns of U matrix contain the basis spectra, and those of the V matrix contain the denaturant dependence of each basis spectrum. Both U and V columns are arranged in terms of their decreasing order of the relative weight of information, as indicated by the magnitude of the singular values in S. The diagonal S matrix contains the singular values that quantify the relative importance of each vector in U and V. An important feature of SVD analysis is that the signal-to-noise ratio is very high in the earliest columns of U and V, and the random noise is mainly accumulated in the latest U and V columns. The wavelength averaged spectral changes induced by increasing denaturant concentrations are represented by the columns of matrix V. Therefore, the plot of the columns of V versus the denaturant concentration provides information about the observed transition.

Urea-induced equilibrium unfolding was analyzed by fitting baseline and transition region data to a two-state linear extrapolation model (20) according to Equation 1, where {Delta}Gunfolding is the free energy change of unfolding for a given denaturant concentration, {Delta}GH2O is the free energy change of unfolding in the absence of denaturant, and m is a slope term that quantitates the change in {Delta}Gunfolding per unit concentration of denaturant, R is the gas constant, T is the absolute temperature, and Kunfolding is the equilibrium constant for unfolding.

(Eq. 1)
The model expresses the signal as a function of denaturant concentration, shown in Equation 2, where yi is the observed signal, yN and yD are the native and denatured baseline intercepts, mN and mD are the native and denatured baseline slopes, [X]i is the denaturant concentration after the ith addition, {Delta}GH2O is the extrapolated free energy of unfolding in the absence of denaturant, m is the slope of a G unfolding versus [X] plot, R is the gas constant, and T is the absolute temperature.

(Eq. 2)
The [urea]0.5 is the denaturant concentration at the midpoint of the transition and, according to Equation 1, is calculated as shown in Equation 3.

(Eq. 3)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of mjSHMT and Molecular Mass Estimation— Purification was performed on 15-g portions of wet bacterial cell paste deriving from 2 liters of culture. A 20-fold purification of the enzyme with a yield of 10 mg per liter of culture was obtained. The purified enzyme exhibited a single band on SDS-PAGE when stained with Coomassie blue and, on analytical ultracentrifugation, sedimented as a single, sharp, and symmetrical peak (see below). mjSHMT, whose predicted subunit size based on the amino acid sequence is 48.2 kDa, eluted in 20 mM sodium phosphate, pH 7.2, from a calibrated size-exclusion chromatography column (Superose 12) with an elution volume corresponding to a protein of 98 kDa, suggesting that the enzyme is a dimer at neutral pH.

Catalytic Properties of mjSHMT—The SHMT activity of the enzyme was assayed at 37 °C, using either H4MPT or H4PteGlu as pteridine substrate. Such a sub-optimal temperature was required by the use of methylene-H4MPT dehydrogenase from M. extorquens AM1 as the coupling enzyme in the assay. Activity, which was measured upon mixing 0.1 µM enzyme with 4 mM L-serine and 50 µM of either pteridine substrates, could only be detected when using H4MPT. The dependence of the steady-state rate of the reaction on substrates concentration conformed to the Michaelis-Menten equation, with best fit values of kinetic parameters as summarized in Table I. The addition of 20 mM magnesium acetate to the reaction mixture did not increase activity, differently from that reported for the enzyme from M. marburgensis (4). The same experiment was repeated under anaerobic conditions, obtaining similar values for the kinetic parameters, and showing that, interestingly, the reaction is not negatively affected by oxygen.


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  		TABLE I
Kinetic parameters of the reactions catalysed by mjSHMT

Kinetic constants are the average of at least three determinations. The range of values was always less than ±10%.

 

Activity could actually be measured also when using H4PteGlu, but only if the enzyme was present in the assay at a much higher concentration (14 µM). In this case, the dependence of the steady-state velocity on the pteridine substrate was linear on a range from 0 to 150 µM. Higher H4PteGlu concentrations could not be employed without incurring problems with the assay. Activity did not change significantly when measured under anaerobic conditions. The specificity constant with H4PteGlu could be calculated, because the observed linear relationship between substrate concentration and initial velocity is evidence that under the conditions of the assay Km» [S], and therefore the Michaelis-Menten equation can be reduced to v0 = kcat/Km·[E0]·[S]. H4PteGlu is known to react spontaneously with formaldehyde (21). Therefore, there was a possibility that the SHMT reaction we observed with H4PteGlu was actually because of the retroaldol cleavage of serine, followed by the spontaneous condensation between the product formaldehyde and the folate to form 5,10-methylene-H4PteGlu. To check this hypothesis, we repeated the experiment under the same conditions but in the absence of H4PteGlu. No formaldehyde could be detected, confirming that the SHMT reaction involving H4PteGlu takes place on the enzyme. The actual binding of H4PteGlu to the enzyme was also demonstrated by the appearance of a typical 490-nm absorbing band, corresponding to a quinonoid intermediate (22), upon mixing 0.2 mM H4PteGlu with 230 µM enzyme and 70 mM glycine at 37 °C in 20 mM potassium phosphate, pH 7.2. Analogously, a quinonoid intermediate was visible after mixing 0.135 mM H4MPT with 14 µM enzyme and 70 mM glycine (data not shown).

The enzyme was also capable of catalyzing the retroaldol cleavage of L-allo-threonine and threo-{beta}-phenylserine to glycine and the corresponding aldehyde (Table I). Addition of 200 mM of either L- or D-alanine to the enzyme resulted in a transamination reaction, as indicated by the decrease of absorbance at 424 nm and the concomitant formation of a new absorption band with a maximum at 324 nm. The 498-nm absorbing band, corresponding to a quinonoid intermediate, which is observed with the E. coli enzyme (16), is absent in the reaction catalyzed by mjSHMT. The kinetics of both reactions, which were carried out at 60 °C, fitted well to the sum of two first-order processes. With D-alanine, the rapid phase exhibited a k = 0.42 min–1 and was responsible for about 3/4 of the total absorbance change, whereas the slow phase had a k = 0.054 min–1. With L-alanine, the fast phase corresponded to a k = 0.092 min–1 and 1/3 of the total amplitude; the slow phase had a k = 0.001 min–1. Although eSHMT transaminates both alanine enantiomers with single first order kinetics, the reaction of D-alanine is also faster with this enzyme (k is 0.038 min–1 with D-alanine and 0.014 min–1 with L-alanine).

Temperature Dependence of Enzyme Activity—The temperature dependence of the rate of retroaldol cleavage of L-allo-threonine was determined, using either eSHMT or mjSHMT as catalyst, over the range from 25 to 85 °C (Fig. 2A). The steady-state velocity of the reaction was measured both under virtually saturating conditions (25 mM substrate) and at about 4% of saturation (0.055 mM substrate). The reaction catalyzed by mjSHMT was found to have an optimal temperature 20–25 °C higher than that catalyzed by the E. coli enzyme. Saturation with the substrate had a stabilizing effect on the activity of both enzymes. Data were used in a global fit to the Arrhenius equation in which the activation energy (estimated to be equal to 70.00 ± 2.10 kJ/mol) was a shared parameter (Fig. 2B).



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  		FIG. 2.
Temperature dependence of enzyme activity. A, the activity of mjSHMT (triangles) and eSHMT (circles) in the retroaldol cleavage of L-allo-threonine was assayed at various temperatures. The steady-state velocity of the reaction was measured either under virtually saturating conditions (25 mM substrate; closed symbols) or at about 4% of saturation (0.055 mM substrate; open symbols). Measurements were carried out over the first minute from the beginning of the reaction, during which linearity was always observed. B, Arrhenius plot for the apparent catalytic constant of the reaction.

 

Urea-induced Unfolding Equilibrium—The equilibrium stability of mjSHMT was studied by monitoring urea-induced unfolding transitions. At neutral pH, in the presence of 10 M urea, 42% residual ellipticity at 222 nm was still present after 24 h at 20 °C, thus indicating that the enzyme structure was not abolished (data not shown). No further changes were observed after 48 h. These results prompted us to explore the effect of urea at acidic pH, with the aim of finding the conditions required for complete and reversible unfolding transitions. At pH 3.0 a single monomeric species is present, as indicated by analytical ultracentrifugation experiments, with a secondary structure content similar to that of the enzyme at pH 7.2 (see below). These observations encouraged us to analyze the effect of urea on mjSHMT at pH 3.0. Increasing urea concentrations (0–10 M) induced structural changes on the enzyme, as revealed by far-UV-CD spectra, and above 6 M the dichroic activity was reduced significantly with a transition midpoint at 2.56 M (Fig. 3). The process was completely reversible, as indicated by the recovery of the dichroic activity after dilution of the denaturant, with {Delta}G and m values corresponding to 13.43 kJ/mol and 5.23 kJ/mol/M, respectively. The ellipticity changes at 222 nm induced by increasing concentrations of urea were analyzed after removal of the high-frequency noise and the low-frequency random error by SVD. The global changes in the spectral region from 210 to 250 nm were analyzed by SVD, which indicates that only two spectral components contribute to the far-UV-CD spectra. The most significant singular values are 1.7 x 105, 0.2 x 105, and 0.03 x 105. All the other singular values are well below 10% of the largest singular value and progressively decrease approaching to zero. A plot of the first and the second columns of the V matrix (V1 and V2) as a function of urea concentration (data not shown) shows transition profiles comparable with those observed by monitoring the 222-nm ellipticity changes.



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  		FIG. 3.
Urea-induced unfolding/refolding equilibrium of M. jannaschii SHMT. Far UV-CD spectra were recorded after 24 h of incubation in 20 mM formate, pH 3.0, at 20 °C at the indicated urea concentrations, at a protein concentration of 0.12 mg/ml. The solid line results from a non-linear regression analysis using Equation 2. Reversibility points ({circ}) were not included in the non-linear regression analysis.

 

Spectroscopic and Analytical Ultracentrifugation Analyses— The secondary, tertiary, and quaternary arrangement of the enzyme at pH 7.2 was investigated and compared with that at pH 3.0, with the aim of detecting possible structural changes caused by the low pH necessary to reversibly denature the enzyme. The far-UV-CD spectrum at pH 7.2 is typical of a predominantly {alpha}-helix protein (Fig. 4A). At pH 3.0, the far-UV-CD spectrum of the holoenzyme is characterized by a profile similar to that at pH 7.2 with the same zero intercept and {Theta}222/{Theta}208 ratio and a slight general decrease of ellipticity compared with that at neutral pH (Fig. 4A). In the near-UV region, the dichroic activity of the enzyme at pH 3.0 is notably decreased in comparison with the enzyme at pH 7.2, and the 289-nm Trp band is absent (Fig. 4B). At pH 3.0, the 420- and 325-nm bands, present in the UV-visible CD spectrum of the holoenzyme at neutral pH and attributable to the PLP cofactor, are absent (Fig. 4C). The UV-visible absorption spectrum at pH 3.0 shows a 3-fold decrease of the absorbance at 420 nm, when compared with that of the holoenzyme at pH 7.2 (Fig. 4D).



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  		FIG. 4.
Spectral properties of M. jannaschii SHMT. A, far-UV-CD spectra were recorded in a 0.1-cm quartz cuvette at 0.13 mg/ml protein concentration. B and C, near-UV and visible-CD spectra were recorded in a 1-cm quartz cuvette at 1.2 mg/ml protein concentration. D, UV-visible spectra were recorded at 2.0 mg/ml. E, fluorescence emission spectra were recorded at 0.13 mg/ml protein concentration (295-nm excitation wavelength). mjSHMT in 20 mM sodium phosphate, pH 7.2, is represented by solid lines and in 20 mM sodium formate, pH 3.0, is represented by dotted lines. All the spectra were recorded at 20 °C after 24 h incubation of the protein at the indicated pH values in the presence of 200 µM DTT and 100 µM EDTA.

 

The intrinsic fluorescence emission spectra of the holoenzyme at pH 7.2 and 3.0, measured upon excitation at 295 nm, are similar and show the same maximum emission wavelength at 347 nm (Fig. 4E). At pH 7.2, as observed with other PLP enzymes in the holo-form (23–25), excitation at either 320 or 418 nm yields fluorescence emission spectra with maxima centered, respectively, at 386 nm and 494 nm (Fig. 5, A and B). A faint energy transfer band at about 510 nm is observed when exciting at 285 nm (Fig. 5A, inset). At pH 3.0, the fluorescence emission spectra of the holoenzyme (Fig. 5, A and B) excited at 320 and 418 nm are centered at the same maximum emission wavelength, although a dramatic decrease of the relative fluorescence is observed upon excitation at 418 nm. At pH 3.0, the 510-nm energy transfer band is absent (Fig. 5A, inset).



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  		FIG. 5.
Fluorescence emission spectra of M. jannaschii SHMT. Spectra were recorded at 2.7 mg/ml protein concentration with the excitation wavelength set at 320 nm (A) and 418 nm (B) in 20 mM sodium phosphate, pH 7.2 (solid lines), and in 20 mM formate, pH 3.0 (dotted lines). The inset shows the fluorescence emission spectrum monitored at 0.2 mg/ml upon excitation at 285 nm. All the spectra were recorded at 20 °C.

 

The relative content of the secondary structure elements in solution, estimated by SELCON 2 (26) using the software DICROPROT v2.5 on the far-UV-CD spectra, at pH 7.2 and 3.0, was at least 98% and corresponded to 36 and 28% {alpha}–helix, 14 and 18% {beta}-sheet, 22 and 24% turns, 8% polyproline II, and 20% of other structures, respectively. The relative amount of {alpha} and {beta} structures was also consistent with that determined by K2D program (27) and SELCON 3 (28). These results indicate that at pH 3.0 the SHMT relative secondary structure composition is closely similar to that at pH 7.2. Secondary structure prediction according to Ref. 29 indicates 50% {alpha}-helix, 16% extended, and 34% loops.

The spectral properties of the monomer at pH 3.0 indicate that the dissociation of the dimer into monomers is accompanied by remarkable changes of the tertiary contacts, as indicated by the near-UV and visible CD signals, and suggest that the secondary structure content of the monomer is similar to that of the dimeric enzyme.

Sedimentation velocity experiments yielded an s20,w value of 6.2 for the enzyme at pH 7.0 and 3.9–4.3 at pH 3.0. Such values correspond to molecular masses of 100 kDa and 50–55 kDa, respectively, for proteins with a spherical shape (30). Equilibrium sedimentation experiments confirmed such results, yielding molecular mass values of 53 ± 4 kDa at pH 3.0 and 80 ± 10 kDa at pH 7.2, when data were fitted to a single species. A very poor fit was obtained for a monomer-dimer association-dissociation scheme at both pH values. Taken together, the performed analyses strongly indicate that the protein is mainly dimeric at neutral pH and dissociates almost entirely into monomers at pH 3.0.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SHMT has been isolated from several sources and extensively characterized, from both the structural and functional points of view (31). Crystal structures of the enzyme from five different eubacterial and eukaryotic sources, either in the unliganded form or in complex with amino substrates and H4PteGlu analogues, have been solved (32–36). Therefore, a clear picture of the amino acid residues which form the active site and are involved in cofactor and substrate binding is available. A recent, theoretical analysis on the structural features of archaeal SHMTs proposed that the environment of the PLP cofactor in these enzymes exhibits no significant differences with respect to their bacterial and eukaryotic counterparts (3). SHMT is characterized by very broad reaction and substrate specificities. In addition to its physiological hydroxymethyltransferase activity, this enzyme is able to catalyze, in the absence of the pteridine substrate, a number of different reactions such as the reatroaldol cleavage of {beta}-hydroxyamino acids and the transamination of both alanine enantiomers (31). Our results show that the catalytic properties of mjSHMT match those of the enzyme purified from other sources and therefore confirm that the PLP catalytic apparatus of SHMT is substantially conserved throughout the kingdoms. A multiple sequence alignment of eubacterial and archaeal SHMTs (the numbering system is based on the amino acid sequence of eSHMT) shows indeed that all residues interacting with PLP are invariant (Fig. 6): His126 and Ala202, the residues that stack, respectively, to the re face and si face of PLP, His129 and Asn102, which appear to stabilize the crucial interaction between Asp200 and the pyridinium nitrogen of PLP, and His203 and Ser 175, which interact with the phenol oxygen of PLP pyridine ring. The residues involved in the binding of the amino acid substrates are also invariant: Glu57, and Tyr65, (the apostrophe indicates that the residues are contributed from the other subunit), which appear to interact with the hydroxyl group of the PLP-bound serine substrate, and Arg363 and Ser35, which bind to the {alpha}-carboxylate of the substrates. On the other hand, the selectivity of mjSHMT toward H4MPT reflects the presence of a specific binding site of the pteridine substrate and clearly represents a distinguishing feature. Interestingly, H4PteGlu can also bind to mjSHMT and take part to the hydroxymethyltransferase reaction, although 450-fold less efficiently than H4MPT. This suggests that significant differences between H4MPT-dependent and H4PteGlu-dependent SHMTs may be limited to only the pteridine binding site. Crystallographic studies have shown that the pteridine substrate binds to the enzyme from different eubacterial and eukaryotic sources with similar modalities, although the stoichiometry and subunit occupancy for the binding is different for each of the four structures solved so far (34, 35, 36, 38). Asn347, which binds to N-1 and N-8 of H4PteGlu, is probably the most important structural element in the pteridine ring recognition. Another important interaction is made by Tyr64, which stacks to the p-aminobenzoic acid ring of H4PteGlu. Other interactions are made by the backbone carbonyls of residues 121, 125, and 127. The portion of the active site that binds the pteridine ring, i.e. the moiety of the molecule that is similar in all different pteridine substrates, is expected to be structurally similar in all SHMTs. Asn347 is indeed an invariant residue (Fig. 6). Tyr64 is present in all aligned sequences except those corresponding to SHMT from methanogens, which have a Leu residue at this position. However, this difference does not seem to be related to the nature of the pteridine substrate, because Pyrococcus SHMTs use a modified folate whose pteridine ring is identical to that of H4MPT (2). Identification of the residues responsible for the pteridine substrate specificity is actually difficult. Apparent differences in the primary structure of SHMTs from methanogens that use H4MPT (note that Methanosarcina SHMT is an exception in methanogens, because it is H4PteGlu-dependent (39)) with respect to the eubacterial and eukaryotic enzymes (Fig. 6) may be related to the particular phylogenetic history of these organisms, rather than to a specific function. Moreover, the available structures of SHMT in complex with H4PteGlu derivatives do not give any clue of how the distinctive portion of the pteridine substrates may bind to the enzyme, and modeling studies did not locate structural elements involved in this specific binding (3).



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  		FIG. 6.
Multiple-sequence alignment of SHMTs from eubacterial and archaeal organisms. The multiple-sequence alignment was obtained employing the T-Coffee server (37) at www.ch.embnet.org/software/TCoffee.html. Dashes represent insertions or deletions. Numbers above the sequences are relative to the sequence numbering of eSHMT. Amino acid one-letter code is used. The invariant residues are displayed in gray boxes. Residues that appear to be invariant only in methanogens SHMTs that use H4MPT are in boldface and are indicated by a circle above the sequences ({circ}). The name of the microorganisms selected for the sequence alignment and their SwissProt accession numbers are as follows: Mja, M. jannaschii Q58992 [GenBank] ; Mth, M. thermoautotrophicum O27433 [GenBank] ; Mma, M. marburgensis P50436 [GenBank] ; Mka, Methanopyrus kandleri Q8TZ19; Mac, Methanosarcina acetivorans Q8TK94; Mmz, Methanosarcina mazei Q8PZQ0; Pab, Pyrococcus abyssi Q9V1B2; Pfu, Pyrococcus furiosus Q8U039; Pho, Pyrococcus horikoshii O59347 [GenBank] ; Aae, Aquifex aeolicus O66776 [GenBank] ; Tma, Thermotoga maritima Q9WZH9; Eco, Escherichia coli P00477 [GenBank] .

 

M. jannaschii is an obligate anaerobe and therefore it is expected that oxygen might affect the hydroxymethyltransferase reaction catalyzed by mjSHMT. We tested the effect of oxygen by performing kinetic studies under both aerobic and anaerobic conditions and using either H4MPT or H4PteGlu as pteridine substrate. Surprisingly, the presence of oxygen does not seem to affect significantly the kinetic parameters of the reaction.

The temperature dependence of the enzyme activity in the allo-threonine aldolase reaction shows that with either mjSHMT or eSHMT the saturation with substrate has a stabilizing effect (Fig. 2A). This is in agreement with previously reported studies on the interactions of substrates and substrate analogues with cytosolic rabbit SHMT, which suggested that the binding of {beta}-hydroxyamino acids results in a conformational change of the enzyme (40). The activation energy of the allo-threonine aldolase reaction calculated from the Arrhenius plots appears to be the same for both enzymes. Taken together, these results confirm that mjSHMT is quite similar in several respects to the enzyme from mesophilic sources.

Notably, mjSHMT is fairly resistant to denaturation. At pH 3.0, the protein shows a global secondary structure content similar to the native dimeric form with a significant perturbation of the tertiary structure, as revealed by the near-UV and visible CD spectra and the PLP fluorescence emission spectrum, accompanied by loss of the dimeric assembly. This finding may indicate that electrostatic interactions play a significant role in the stabilization of the dimer. The {Delta}G of the monomer secondary structure unfolding is below the lowest limit reported for many globular proteins from mesophiles, whereas the transition midpoint is at 2.56 M denaturant, a value similar to that reported for the dimeric E. coli enzyme at neutral pH (41). This fact indicates that the monomeric protein can tolerate high concentrations of denaturant without showing a remarkable conformational stability at 20 °C. Interestingly, the notable resistance of the enzyme against urea-induced denaturation is shown by the persistence of a significant amount of secondary structure in the presence of 10 M urea at pH 7.2. Therefore, the monomeric state of mjSHMT at pH 3.0 should be considered a protein resistant against urea-induced denaturation rather than a stable protein (42).

A recent structural comparison between SHMT from mesophilic and thermophilic sources, carried out through the application of homology modeling, indicated that the thermal stability in SHMT may be the result of the combination of an increased number of charged residues at the protein surface and an increased hydrophobicity at the protein core (3). The experimental results obtained with mjSHMT agree with these hypotheses.


   FOOTNOTES
 
* This work was supported by the Italian Ministero dell'Istruzione, dell'Università, and della Ricerca. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dipartimento di Scienze Biochimiche, Università degli Studi di Roma "La Sapienza," Piazzale Aldo Moro 5, Roma 00185, Italy. Tel.: 39-06-49917569; Fax: 39-06-49917566; E-mail: roberto.contestabile{at}uniroma1.it.

1 The abbreviations used are: SHMT, serine hydroxymethyltransferase; mjSHMT, M. jannaschii SHMT; eSHMT, E. coli SHMT; H4MPT, tetrahydromethanopterin; H4PteGlu, tetrahydropteroylglutamate or tetrahydrofolate; DTT, dithiothreitol; PLP, pyridoxal phosphate; SVD, singular value decomposition algorithm. Back


   ACKNOWLEDGMENTS
 
We thank Professors Verne Schirch and Rolf Thauer for help during the writing of the manuscript and for helpful discussions. We are grateful to Eprova AG, Schaffhausen, Switzerland, for kindly providing pure (6S) H4PteGlu.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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