THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 248, No. 2, Issue of January 25, pp. 403-408, 1973

Printed in C.S.A.

Purification and Characterization of Thermostable 5, lo- Methylenetetrahydrofolate Dehydrogenase from 5Clostridium thermoaceticum*

(Beceived for publication, August 14, 1972)

WILLIAM E. O’BRIEN,~ JOHN M. BREWER, AND LARS G. LJUNGDAHL

From the Department of Biochemistry, University of Georgia, Athens, Georgia $0601

SUMMARY

Methylenetetrahydrofolate dehydrogenase (5, lo-methyl- enetetrahydrofolate: NADP oxidoreductase EC 1.5.1.5) has been purified from Clostridium thermoaceticum, an obligate anaerobic thermophile. The enzyme has a molecu- lar weight of 55,000 f 5,000 and consists of 2 subunits of equal molecular weight. The sedimentation constant s&, is 3.8 S, the partial specific volume 0.752 cc per g, and the Stokes radius 31.7 A. The enzyme appears homogeneous during Sephadex chromatography and uitracentrifugation, but during disc gel electrophoresis at pH 8.9 two bands of equal intensity are seen. Both bands are enzymatically active, and, when isolated individually, they again show the same two bands in the electrophoresis, indicating an equilib- rium between the two forms. The enzyme has high thermal stability with a temperature optimum above 64” and exhibits a broken line in an Arrhenius graph. The apparent K, for NADP is 9 X lo-” M and for methylenetetrahydrofolate 3.5 X lo-5 M. The enzyme is specific for NADP and NAD is a competitive inhibitor with a K; of 2 X 10V4 M.

Methylenetetrahydrofolate dehydrogenase (5, lo-methylene- tetrahpdrofolatc :NADP osidoreductsse, EC 1.5.1.5) catalyzes the oxidation of met,hylcnetetrahydrofolate by NADP to meth- enyltetrahydrofolate as follows:

Methylene-THF + N.4DPc ti

methenyl-THF + NAI)PH + H+

Closfridium tmer,,loc~~~fi~wn, which is an obligative thermophilic anaerobe, ut.ilizcs CO4 as a terminal electron acceptor and syn- thesizes n&ate rle yr,o1.0210 from COe. It has been demonstrated

* This work was supported by Grants AM 12913 from the Na- t.ional Inst,itut.es of Health and GB 13031 from the National Science Foundation. The paper is from a dissertation submitted by W. 11. O’Brien in pa.rtial fulfillment of the requirements for the Ph.D. degree. A portion of this work was reported at the 62nd Bnnual Meeting of the American Societ,y of Biological Chemists in San Francisco, 1971 (1).

$ Present address, Departments of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.

that in the formation of the methyl group of acetate, COZ is first reduced to formate and then to 5-methyltetrahydrofolate. The methyl group of the latter is transferred to a corrinoid to form a methylcorrinoid from which acetate is formed by carboxylation (24). This investigation was initiated to establish the presence in C. themnoaceficum of methylenetetrahydrofolate dehydrogen- ase, one of the enzymes essential for the formation of methyltetra- hydrofolate from forma.te and consequently also for the synthesis of acetate from COZ in C. fhermoaceticum. The purification and characterization of the enzyme is also a part of our studies regard- ing enzymes which function optimally at elevated temperatures. Like other enzymes from C. thermoaceticum (5), the methylene- tetrahydrofolate dehydrogenase has a temperature optimum con- siderably higher than that of corresponding enzymes isolated from mesophilic organisms.

EXPERIMENTAL PROCEDURES

Cell Material-C. thermoaceticum was grown as described by Ljungdahl et at. (5). The cells could be stored at - 15” for more than 4 years without loss of methylenetetrahydrofolate dehydro- genase activity.

Enzyme &say->Iet,hylenetetrahydrofolate dehydrogenase was assayed as described by O’Brien and Ljungdahl (6) except bhat NADP was used instead of NAD. A unit of the enzyme is the amount required to form 1 pmole each of 5, IO-methenyltetra- hydrofolate and NADPH per min at 37” and the specific activity is expressed in units per mg of protein. These products absorb at 356 urn with a combined extinction coefficient of t = 29.7 x 1Oa cm-’ ~1-1 when present in equal concentrations. The reac- tion was followed with a Gilford model 2000 recording spectro- photometer.

Analytical *llethods--Protein was determined either by the biuret metJlod (7) or by the method of Warburg and Christian (8). hnalytical and preparative disc gel elcctrophoreses in acrylamide gels were performed as described by Brewer and Ash- worth (9). Electrophoresis in polyacrylamidc gels containing sodium dodecyl sulfate was done as described by Weber and Os- born (10). Sediment,ation velocity analyses and sedimentation equilibrium were performed as described by Schachman (11) and Yphant,is (12), respectively, with a Spinco model E ultra- centrifuge fitted for schlieren and interference optics. Calcula- tions of ammonium sulfate concentrations were done according to DiJeso (13). To determine t,he amino acid composition the

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enzyme was first dialyzed against distilled water and then con- centrated HCl was added to obtain a 6 ~\i concentration. The hydrolysis was performed under vacuum at 110” for 24,48, and 72 hours and about 4 nmoles of the enzyme were used in each hydrol- ysis, The a.mino acids were analyzed in a Beckman model I2OC automatic amino acid analyzer.

Chemicals-NADP, NAD, FAD, FMN, and folic acid were ob- tained from Sigma. Ammonium sulfate was the enzyme grade quality from Schwarz-Mann. Diethylaminoethylcellulose (DE 23) was obtained from Whatman, and Sephadex was from Phar- macia. 5, IO-Methylenetetrahydrofolate was prepared from tetrahydrofolate and formaldehyde as described earlier (6). NADP and 5,10-methylenetetrahydrofolate concentrations were determined with methylenetetrahydrofolate dehydrogenase. Yeast enolase was prepared according to Westhead and McLain (14), porcine crythrocyte carbonic anhydrase B (15) was a gift from Dr. R. B. Ashworth of our department, and catalase was obtained from Sigma.

Purification of Methyle~fetrahydrofolafe Dehydrogenase

All steps were conducted at O-4” at pH 7.0 in either potassium maleate or potassium phosphate buffers. The purification is summarized in Table I.

Step I. Preparation of Cell-j?ee Extracts-Frozen cells (160 g) of C. thermoaceticum were suspended in 480 ml of 0.05 M potassium maleate buffer and passed through a French pressure cell (Ameri- can Instrument Company) at 13,500 p.s.i. The resulting ma- terial was centrifuged at 37,000 x g for 1 hour. The supernatant liquid was decanted and the pellet washed with additional buffer and centrifuged again. The two supernatant liquids were com- bined.

Step II. Ammonium Sulfate Fractionation-.4mmonium sulfate was added to a final concentration of 40% saturation. The pH fell to 5.3 upon addition of the ammonium sulfate. The solution was stirred for 30 min and then the precipitate was removed by centrifugation. The pellet was washed with 40% ammonium sulfate and centrifuged again. The two supernatant solutions were combined. Solid ammonium sulfate was added to the supernatant solutions to give a final concentration of 600% satura- tion. The solution was stirred for 30 min and the precipitate containing the enzyme was collected by centrifugation at 18,000 X g for 30 min.

Step III. Chromatography on Sephaclex G-200-The precipitate from the 40 to 60% ammonium sulfate cut was taken up in 0.10 M potassium phosphate buffer (80 ml) and loaded onto a column (10 x 100 cm) of Sephadex G-200 equilibrated with the same buffer. Fractions of approximately 22 ml each were collected.

TABLE I Purification of methylenetetrahydrofolate dehydrogenase

from 160 g of frozen cells of C. lhermoaceticum

step Total units

I. Cell-free extract.. 19,250 II. (NH&SO., precipitate: 40 to 600%

fraction, pH 5.3 3,900 III. Sephadex G-200. 475 IV. DEAE-cellulose. 10

V. (NH&Sod-back extraction: 55%,, pH 7.0.. . 4

35,300

31,200 7.5 9,900 21 1,750 172

Specific activity

units/mg

1.7

362

Fractions containing enzyme of specific activity above 10 were pooled. The enzyme was precipitated by addition of solid am- monium sulfate to 80% saturation and recovered by ccntrifu- gation.

Step IV. Chromatography on DEAE-Cellulose-The precipitate from the previous step was dissolved in 20 to 30 ml of distilled water and dialyzed against four l-liter changes of 0.001 M potas- sium phosphate, pH 7.0. The dialyzed material was loaded onto a DEAE-cellulose column (2.5 to 30 cm) which had been equili- brated with potassium phosphate buffer, pH 7.0, and then washed with distilled water. The protein was elut,ed in fractions of 5 ml with a l-liter linear gradient of 0.001 M to 0.10 M potassium phos- phate buffer. The fractions containing the enzyme were pooled and the enzyme was precipitated by addition of ammonium sul- fate to 80% saturation.

Step V. Ammonium Sulfate Back Extraction-The precipitate from the previous step was washed with ammonium sulfate solu- tions of 65% saturation. It was then dissolved by extraction with approximately 20 ml of a solution of ammonium sulfate at 55% saturation. The ammonium sulfate solutions were ad- justed to pH 7.0 with 4 N NH40H. The 65% solutions and the residue after the 55yo extraction had very little activity.

RESULTS

Physical Properties and Purity of Xethylenetetrahydrofolate Dehydrogenase from C. thermoaceticum

Electrophoresia-Polyacrylamide gel elect,rophoresis of meth- ylenetetrahydrofolate dehydrogenase of specific activity 362 routinely showed two bands migrating closely together with ex- tensive smearing. To determine whether this was due to oontam- ination or to an artifact of the electrophoresis experiment, prepar- ative disc electrophoresis was employed on material of specific activity of about 250. Fig. 1 shows the activity profile obtained from such an experiment. Two peaks of activity were always observed but were never completely separated. As a control a purified preparation of the enzyme from Clostridium formico- aceticum (19) was also run in a separate experiment and the profile is included in Fig. 1.

Peaks A and B were separately pooled and precipitated by ad- dition of solid ammonium sulfate to 80% saturation and dialyzed against 0.1 M potassium phosphate buffer, pH 7.0. Peak ,4 con- tained a yellow contaminating protein and had a specific activity of 268. Peak B had a specific activity of 362. Both fractions were subjected to analytical gel electrophoresis. Fig. 2 shows the results of this experiment. Numerous minor bands are evi- dent in the gel of fraction A, as expected from the lower specific activity and the presence of color. However, important is the fact that both the A and B fractions again show the presence of two major bands migrating close together with the extensive smearing always encountered with this protein. It is our conten- tion that the enzyme is near purity at a specific activity of 362.

Amino Acid Composition of Methylenetefrahydrofolak Dehydro- genase from C. thermoaceticum-The amino acid composition of two different preparations of the enzyme of a specific activity of 362 is given in Table II. The results are corrected for 100% recovery of amino acid residues. The variability encountered in the compositions is probably due to the small amounts of ma- terial analyzed. From the amino acid composition (17) the par- tial specific volume of the enzyme was calculated to be 0.752 cc per g.

Molecular Weight and &%&units-The molecular weight of preparations of enzyme of 362 specific activity was measured by

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FORMICOACETICUM .’ --

60 80 100 120 140

FRACTION NUHBER

FIG. 1. Activity profiles obtained from preparative disc elec- trophoresis of methylenetetrahydrofolate dehydrogenase from Clostridium thermoaceticum and Clostridium formicoaceticum. Approximately 10 mg of enzymes containing 2500 units were applied to the gels in both experiments. The disc electrophoresis system was at pI1 9.0 with 7.5% acrylamide gel at 0” and with 45 ma applied (9). Fractions of 5 ml were collected at intervals of 20 min. After concentration and dialysis of the enzyme from C. thermoaceticum Peak A had a specific activity of 2G8 and Peak R of 361.

Yphantis sedimentation equilibrium centrifugation and by equi- librium centrifugation at low speed. In the latter experiment, protein concentrations were measured with a monochromator- scanner. Based on the amino acid composition, the partial specific volume of the protein is 0.752 cc per g. In the case of the Yphantis experiment, me used a rotor speed of 28,400 rpm, a pro- tein concentration of 0.5 mg per ml, a rotor temperature of 4”, and 0.1 M potassium phosphate, pH 7.0, as the solvent. After 24 hours, the slope of the log concentration versus radius squared plot was 0.869, which corresponds to a molecular weight of 60,000.

The low speed equilibrium experiment gave a molecular weight of 50,000. The dependence of logarithm of protein concentra- tion upon radial distance squared was linear in both cases (the correlation coefficient in the case of the Yphantis experiment was 0.99978)) which supports our contention that the protein prepara- tions were homogeneous.

Polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate was performed on the enzyme in the presence of proteins of known molecular weight after treatment with 1 y0 sodium do- decyl sulfate and 0.1 M mercaptoethanol (10). The enzyme mi- grated with porcine erythrocyte carbonic anhydrase B of mo- lecular weight 30,000 (15). The reliability of molecular weight obtained by the electrophoresis in sodium dodecyl sulfate is esti- mated to be +~10% (10). The data suggest that 5, lo-methyl- enetetrahydrofolate dehydrogenase from C. thermoaceticum has a molecular weight of about 55,000 and is composed of 2 subunits of equal molecular weights.

Sedimentation Constant-The sedimentation constant of the

FIG. 2. Analyt’ical disc electrophoresis of Fractions A and B from preparative disc electrophoresis of met)hylenetetrahydro- folate dehydrogenase from Clostridium thermoaceticztm. The pH 9.0 system with 7.5oj, acrylamide gel was used (9). Staining was with Amido black in 7yo acetic acid. The set on the left was with about 50 fig and that to the right with 100 pg of protein. The two sets are from different preparations of the enzyme.

TABLE II

Amino acid composition of methylenetetrahydrofolate dehydrogenase from C. thermoaceticuma

Amino acid Experiment 1 Experiment 2 Average

Tryptophan.. -b -b -b . Lysine 39 32 36 Histidine. 14 9 12 Arginine 21 19 20

Aspartic acid. Threonine. Serine Glutamic acid.. Proline. Glycine . Alanine.

43 46 45 16 20 18 16 19 18 45 51 48 26 30 28 -b 45 45 57 58 58

Half-cystine . . Valine Methionine . . . Isoleucine. . . Leucine. . . . . . . Tyrosine....... . . . . Phenylalanine. .

6 9 8 64 51 58

G 9 8 40 39 40 42 43 43 3 5 4 6 7 7

-

a Calculated for a molecular weight of 55,000 assuming 100% recovery.

b Present but not quantified.

enzyme was determined in partially purified preparations by su- crose density gradient centrifugation (18) with bovine erythro- cyte carbonic anhydrase B, yeast enolase, and horse liver catalase as st,andards. A value of 3.8 + 0.2 S was obtained. The sedi- mentation constant was also determined on an enzyme prep- aration of 362 specific activity by measurement of the rate of movement of the protein-solvent boundary with a photoelectric scanner. The protein preparation had an absorbance at 280 nm

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!- 60 100 140

FRACTION

FIG. 3. Determination of the Stokes radius of methylenetetra- hydrofolate dehydrogenase from CIoslridium thermoaceticum. Approximately 0.5 mg each of methylenetetrahydrofolate de- hydrogenase (Peak III), enolase (II), and carbonic anhydrase (IV), in a volume of 1 ml was added to a Sephadex G-100 column (1.5 x 90 cm). Fractions of 0.8 ml were collected. The solvent, was 0.1 M potassium phosphate, pH 7.0. Blue dextran (I) and vitamin Blz (V) were passed through the column in a second experiment.

of 0.3. Essentially only one boundary was observed with an A value of 3.8. The low protein concentrations used in these ex- periments give values essentially equivalent to siO,u,.

Stokes RadiusTo see whether large amounts of lipid or carbo- hydrate are associated with methylenetetrahydrofolate dehydro- genase from C. thermoaceticum, the Stokes radius was determined by chromatography on a column of Sephadex G-100. The col- umn, 1.5 x 90 cm, was prepared for descending flow with 0.1 M potassium phosphate, pH 7.0, as solvent. Blue destran and vitamin Blz were used to measure the void and total volumes of the column. Yeast enolase and porcine erythrocyte carbonic anhydrase B (15) were used as standards. The Stokes radii of these enzymes are 34.8 A and 24.7 A, respectively. Methylene- tetrahydrofolate dehydrogenase eluted from the column just be- hind enolase (Fig. 3), corresponding to a Stokes radius of 31.5 A

(16). The Stokes radius was also calculated from the molecular

weight sedimentation constant and partial specific volume. With the use of values of 55,000, 3.8 S and 0.752 cc per g, the Stokes radius was calculated as 31.7 A which is in good agree- ment with the measured value. This agreement suggests that no large quantity of nonproteinaceous material is associated with the enzyme.

Enzymatic Properties of 3lethylenetetrahydnofolate Dehydrogenase

from C. thermooceticum

Stability-The purified protein was maintained at 4” at various protein concentrations for at least 2 months without loss of activ- ity and was kept at -20” for at least 2 months at dilute protein concentrations of 0.05 mg per ml or less. If the protein concen- tration was increased above this level irreversible precipitat,ion resulted upon freezing.

Coenzyme Speci$city and K, Values-The enzyme was active only with NADP and a reaction was not observed with NAD, FMN, or FAD. The apparent K, for NADP was 1.9 x 10-d M at 20’ and 37” and 0.9 X 10-a M at 55” at pH 7.0. The K, at pH 7.0 for 5,10-methylenetetrahydrofolate was at 20”, 4.2 x 10e5 M; 37”, 3.5 X 10s5 M; and at 55”, 3.0 X lop5 M. Although NAD was not a substrate it competitively inhibits the reaction as shown in Fig. 4. The apparent K; for NAD is 2 x 10-d M.

40 80 120

1/(NADP+l.104, ,-I

FIG. 4. Competitive inhibition of methylenetetrahydrofolate dehydrogenase from Clostridium fhermoace/icum by NAD. The reactions were performed at 37” in 0.2 x potassium maleate, pH 7.0, containing 0.24 M mercaptoethano!. The concentrat,ion of methylenetetrahydrofolate was 10m3 M. SAD was added as indi- cated. The reactions were initiated by the addition of 0.038 unit, of the enzyme. Total assay volume was 0.25 ml.

L PO 4’0 6’0

TEMPERATURE

FIG. 5. The effect of t,emperature on the reaction rate of meth- ylenetetrahydrofolate dehydrogenase from Clostridium thermo- aceticurn. The concentration used for SADP was 2.4 X 10-S M.

The reactions were initiated by addition of 0.0004 unit of the enzyme. Inset: Arrhenius plot.

Eflect of Temperature on Reaction-The effect, of temperature on the rate of the enzymatic reaction is shown in Fig. 5. The buffer and water used in the assays were maintained at the assay tcm- perature. All components were added to the cuvettes which were previously equilibrated at the desired temperature and a further 5-min thermal equilibration period was allowed before the reaction was initiated by addition of the enzyme. The reac- tions were followed continuously for 30 to 60 s and were linear during that period.

The enzyme could not be assayed at temperatures above 64” due to a rapid nonenzymatic breakdown of the substrate, 5, lo- methylenetetrahydrofolate. Under the conditions of the experi- ment, the activity of the enzyme cont,inued to increase through- out the temperature range of 9-64”. This is not the case for the methylenetetrahydrofolate dehydrogenase isolated from C. formicoaceticum which is a mesophile. The enzyme from this organism has been purified to a specific activity of 428 (19). It was assayed under exactly the same conditions, as described in Fig. 6, and at 45” the enzyme was rapidly inactivated.

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5o" 0

61°

20-

10 20 30

MINUTES

FIG. 6. Heat inactivation of methylenetetrahydrofolate de- hydrogenase from Clostridi~~ thermoaceticum. One milliliter of 0.1 M potassium maleate, pH 7.0, was raised to the temperature indicated and 0.12 unit of methylenetetrahydrofolate dehydro- genase was added. Portions of 0.01 ml were withdrawn on a time schedule and assayed at 37”.

The inset of Fig. 5 shows an Arrhenius plot of the data from the enzyme from C. thermouceticum. The plot gives a broken line with the break at about 35”. An apparent activation energy of 9600 cal per mole below 35’ and 6500 cal per mole above 35” was calculated from the data. The biphasic relationship is not due to a change in apparent Michaelis constants for either NADP or 5,10-methylenetetmhydrofolate since these values are essen- tially identical at 20”, 37”, and 55”, and also the concentration of NADP was 26 times that of the R, at 37” and of 5, lo-methyl- enetetrahydrofolate about 30 times that of the K,. Thus we believe the data shown in Fig. 5 represent maximum velocities at respective temperatures.

Thermal Stability oj Enzyme-To study the thermal stability of methylenetetrahydrofolat,e dehydrogenasc, we incubated the en- zyme in 0.1 M potassium malcatc, pH 7.0 (in the absence of sub- strates), for various temperatures and times and then assayed at 37” in the normal assay mixture. Fig. 6 shows the results of such an invest,igation. .At 50” the enzyme is virtually unaffected by incubation for 30 min. At 61” the enzyme is first inactivated until about 80% of the activity remains, after which no further inactivation takes place. Similarly at 68” there is a rapid de- crease in enzymic activit.y during the first 10 min followed by a plateau at approximately 50% inactivation for the remaining 20 min of the experiment. These data suggest that the enzyme may undergo a conversion iuto a more stable, but less active, form during heating.

DISCUSSION

C. thermoaceticum synthesizes acetate with both carbons de- rived from COz (2). 13y the USC of ‘Glabeled compounds and especially 1KQ in pulse labcling experiments it has been shown t.hat mct.hylcorrinoids (20, 21), methyltetrahydrofolate (3, 4), and formate (20, 22) arc intermediates in the formation of the methyl group of acetatc. Hence it has been postulated that the reduction of CO? proceeds via formate and one carbon de- rivatircs of tetrahydrofolate. Previously formyltetrahydro-

folate synthetase (5) and a NADP-dependent formate dehydro- genase (23) have been found in C. thermoaceticum. The fact that methylenetetrahydrofolnte dehydrogenase is also present in a relatively large amount, in this organism lends additional support to t,he idea that the met,hyl group of acetate is formed from COZ

via the tetrahydrofolate pathway. Further evidence for this pathway in acetate synthesis has been obtained with C. jormico- aceticum . This organism also synthexizcs acetate from Con (24, 25). It contains all enzymes necessary for the conversion of CO* to methyltetrahydrofolate (6) ; furthermore, cell-free extracts of C. jormicoaceticum incorporate the methyl groups of methyltetrahydrofolate and methylcobalamin into acetate (6).

Methylenetetrahydrofolate dehydrogenase has been purified from numerous microbial sources including yeast (26), Esch- erichia coli (27), Salmonella typhimurium (28), Clostridium cylin- drosporum, and Clostridium acidiurici (29). All of these enzymes utilize NADP as the electron carrier. An NAD-linked enzyme has been demonstrated in Ehrlich ascites cells (30) and more recently in C. formicoaceticum (6). The enzyme from C. cylin- drosporum has been purified to homogeneity as judged by disc electrophoresis (29) and it has a specific activity of 95 at 25”. The C. thermoaceticum enzyme, which we believe is pure, has a specific activity of 362 at 37” and at 25” about 210. Thus the latter enzyme is considerably more active and has the highest activity reported for any NADP-dependent methylenetetrahy- drofolate dehydrogenase. However, a still higher specific ac- tivity has been found for the NAD-dependent enzyme from C. jormicoaceticum. This enzyme has been purified to about 707, purity and has at this stage a specific activity of 428 at 37” (1, 19).

Several reports indicate that proteins from thermophilic or- ganisms exhibit higher temperature optima and thermal stabili- ties than corresponding proteins from mesophilic organisms. Numerous theories have been advanced in an attempt to explain thernmophilicitp. These include protective factors within the cell (31), higher average hydrophobicity (31), and high rates of protein turnover (32, 33). In addition, studies with a cy-am- ylase isolated from Bacillus stearothermophilus (34) indicated that this protein may exist as a random coil in solution. How- ever, there is now in the literature ample evidence that supports the conclusion that the inherent thermal stability of proteins isolated from thermophilic bacteria is not due to any external factors or extraordinary structural differences as previously believed but rather to subtle intrinsic differences in the protein structure. Thorough investigations of the glyceraldehyde 3-phosphate dehydrogenase from B. stearothermophilus (35) and the lo-formyltetrahydrofolate synthase from C. thermoaceticum (5, 36) in addition to other proteins (37-43) have verified this conclusion. It uow appears that the thermophilic cr-amylase isolated from B. stearothermophilus is the exception rather than the rule concerning the thermal stability of proteins.

Methylenetetrahydrofolate dehydrogenase from C. thermo- aceticurn, like other cnzymcs from this organism, is exceptionally stable to heat inactivation. The optimum temperature for activity is in excess of 64”, which is snme 20” above the temp- erature optimum for the same enzyme isolated C. formico- aceticum (I). Unfortunately studies regarding thermal stability and temperature optimum of the enzyme from C. cylindrosporum are lacking (29). The enzyme from C. thermoaceticum exhibits a brokeu Arrhenius plot as do several other thermostable proteins (33, 42, 44). Presently we do not know the nature of the change of activation energy for the thermophilic proteius, and its rcla- tion to t,hermal stability is conjectural. However, broken Ar- rhenius plots have not been found in the same enzymes isolated from mesophiles, which suggests a relation to thermophilicity.

During exposure to temperatures above 55” the methyl- enetetrahydrofolate dehydrogenase from C. thermoaeeticum undergoes initially a rapid decrease in activity, but the activity

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stabilizes after its initial decrease at a level which appears de- 7. GORN.\LL, A. G., B.?RDAWILL, C. J., AZND DAVID, M. M. (1949 pendent on the temperature. Thus at 61” about 80% and at J. Biol. Chem.177, 751

68” about 50% of the original activity remains although the 8. W.4RBURG, O., AND CHRISTIAN, W. (1941) Uiochem. 2.310, 384

enzyme is exposed to the elevated temperature for 30 min or 9. BREWER, J. M., AND ASHWORTH, 11. B. (1969) J. Chem. Educ.

46. 41 more. This suggests that the enzyme during heating undergoes a structural change, producing a form which has greater heat stability. This is in agreement with the observation regarding the broken Arrhenius plot which may indicate a conformational change of the protein during heating.

During electrophoresis we observed two active forms of methyl- enetetrahydrofolate dehydrogenase from C. thermoaceticum. The two forms could not be demonstrated in the ultracentrifuge or on chromatography on Sephades. This suggests that the two forms are not formed through association or dissociation of subunits. It is not likely that the two forms are due to the presence or absence of substrate bound to the enzyme as has been demonstrated for methemoglobin reductase (45) since each form after separation gives rise to both forms. A possible ex- planation of the existence of the two forms could be that they are artifacts due to the rather alkaline pH (9.0) during the disc electrophoresis. However, it is of interest that Lazowska and Luzzati (46) recently demonstrated two forms of the methyl- enetetrahydrofolate dehydrogenase from Saccharomyces cerevis- iae by DEAE-cellulose chromatography. Furthermore the en- zyme from C. formicoaceticum exhibits only one form in the same electrophoresis system as used for the enzyme from C. thermoaceticum. Therefore, we suggest that the enzyme exists in two forms and that perhaps these forms are related to the conformations1 changes which wc observed during the tempera- ture studies described above.

Sufficient data have not yet been collected for a thorough comparison between the chemical and physical parameters of the thermophilic methybnetetrahydrofolate dehydrogenase and the same enzyme from mesophilic clostridia. However, the apparent K, values and molecular weight of the t,hermophilic enzyme do not differ much from those of C. cylindrosporum (29) or C. formicoaceticum (1, 19). This indicates that the thermophilic dehydrogenase does not exhibit a drastically unusual protein structure, which would explain its higher thermal stability. Instead our data suggest that as with many other thermophilic enzymes only a small change, e.g. in the amino acid composition or sequence, may account for the higher stability. Further work is needed to elucidate this point.

Acknowledgment-The authors are indebted to Mr. T. E. Spencer who did the ultracentrifuge experiments.

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16. ACKERS, G. K. (1964) Biochemistry 3, 723 17. COHN, E. J., AND EDSALL, J. T. (1943) Proteins, Amirzo Acids

and Peptides, p. 373, Reinhold, New York 18. MARTIN. R. G.. END AMES. B. N. (1961) J. Biol. Chem. 236.

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William E. O'Brien, John M Brewer and Lars G. LjungdahlClostridium thermoaceticum5,10-Methylenetetrahydrofolate Dehydrogenase from

Purification and Characterization of Thermostable

1973, 248:403-408.J. Biol. Chem. 

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